Antibodies for guanylyl cyclase receptors

ABSTRACT

Monoclonal antibodies that act as potentiators, stimulators and agonists of guanylyl cyclase receptors are disclosed.

BACKGROUND OF THE INVENTION

Intracellular signaling via 3′,5′-cyclic guanosine monophosphate (cGMP)plays an important role in many fundamental physiological processesincluding regulation of vascular tone, renal and cardiac function,immune responsiveness, thrombocyte activation, retinal phototransductionand bone growth Because the level of endogenous cGMP modulates theaforementioned biological processes, molecules that regulate cGMPsynthesis serve as natural targets for new drug discovery andtherapeutic development. Guanylyl cyclases are such molecules.

More specifically, cGMP is produced by the catalysis of guanosinetriphosphate (GTP) by one of two families of guanylyl cyclase enzymes:the particulate guanylyl cyclases (pGCs) and the soluble guanylatecyclases (sGCs). The pGCs generally exist as homodimers comprising anextended intracellular domain which includes the catalytic domain,membrane-spanning regions, and an extracellular ligand-binding domain.In contrast, the sGCs are intracellular heterodimeric moleculescontaining a prosthetic heme group which can be activated by nitricoxide. Upon formation of the NO-heme complex, sGCs undergoconformational changes resulting in large increases in catalyticactivity. The cGMP thus produced can regulate a number of effectormolecules including protein kinases, phosphodiesterases and ionchannels.

Of particular relevance to a host of therapeutic indications is thefamily of particulate guanylyl cyclases activated by natriureticpeptides. Natriuretic peptides are cyclic peptide hormones 28-32 aminoacids long which are synthesized as longer preproproteins and processedto yield the mature peptides. Examples of natriuretic peptides include:A-type or atrial natriuretic peptide (ANP), which is released from theheart, urodilatin, the differentially processed form of ANP produced bythe kidney, B-type natriuretic peptide (BNP), which is synthesized inthe ventricular myocardium and C-type natriuretic peptide (CNP), whichis produced by a number of cell types including endothelial cells andchrondrocytes (Potter, et al Endocrine Reviews, 27:47, 2006).

One membrane-bound GC bound by ANP, urodilatin and BNP is thenatriuretic peptide receptor A (also known as NPRA). Activation of NPRAby these hormones leads to a variety of physiological responsesincluding vasorelaxation, natriuresis, diuresis, lipolysis, inhibitionof cardiac hypertrophy and ventricular fibrosis, inhibition of therenin-angiotensin aldosterone system, and inhibition of sympatheticnerve activity. CNP, on the other hand, serves as a potent agonist fornatriuretic peptide receptor B (NPRB). CNP-dependent activation of NPRBcan also lead to vasodilation as well as stimulation of long bonegrowth. All of the natriuretic peptides have very short plasma halflives ranging from approximately 2-20 minutes. One reason for theirrapid turnover is that they are degraded by proteases such as neutralendopeptidase (NEP), meprin A and dipeptidyl peptidase IV. Moreover,ANP, urodilatin, BNP and CNP also bind to the non-guanylyl cyclaseclearance receptor, natriuretic peptide receptor C (NPRC). Binding toNPRC results in internalization of the peptides subsequent lysosomaldegradation. See, e.g., Cohen et al., J. Biol. Chem., 271:9863 1996.

NPRA has been shown to play an important role in the regulation ofcardiorenal function. Activation of this receptor by ANP and BNP leadsto a reduction in cardiac filling pressures, decrease in afterload,diuresis and natiuresis and inhibition of sympathetic and neurohormonalsystems such as the renin-angiotensin-aldosterone pathway. Extended NPRAactivation has cardiac antihypertropic and anti-fibrotic effects. ANPand BNP are produced by the heart in response to stress and stretch andare elevated in patients with heart failure. NPRA contains anintracellular GC domain and exerts its effects through the production ofcGMP. However, as with many single transmembrane hormone receptorsidentification of small molecule agonists using conventional approacheshave not been successful.

Recombinant forms of NPRA ligands have been approved for treatment ofacute decompensated heart failure. However, these recombinant ligandshave very short half-lives (typically 20 minutes or less) and thus mustbe administered by extended IV infusion. The recombinant form of BNP(Nesiritide) was approved in the U.S. in 2001 for the treatment of acutedecompensated heart failure. Recombinant human ANP (Carperitide) wasapproved in Japan in 1995 for the same indication and recombinanturodilatin (Ularitide, renal form of ANP) is currently in clinicaltrials. Review of Nesiritide data submitted to the FDA during theapproval process has further revealed significant safety concerns moreparticularly, increased mortality and reduced renal function, with theadministration of recombinant BNP. While many of these safety concernshave subsequently been thought to be speculative, there remains aquestion as to the potential of the recombinant natiuretic peptides toinduce adverse events. As such, the use of the currently approvedcompositions has been limited to an acute indication and there remains aneed for additional therapies targeting the NPRA pathway for managingheart failure in a more chronic setting. A therapeutic interventionleading to the activation of the NPRA pathway that would allow for thetreatment of both acute decompensated heart failure with a singleadministration and chronic heart failure with weekly or monthlyinjections would greatly benefit patients suffering from these seriousconditions. To date such a therapy remains to be found.

Although efforts to modify natriuretic peptides and/or antagonize GCreceptors have yielded some therapeutic compositions for use in acutedecompensated heart failure, these efforts have failed to produce arobust therapeutic application for chronic heart failure. Thus, there isa current and continuing need to develop molecules that regulate pGCs,which are safe, have superior stability in vivo and effectively modulatethe activation of the natriuretic peptide system.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention addresses the above-identified needthrough the use of novel antibodies which act as potentiators orstimulators of guanylyl cyclase receptors. Specifically, the inventionrelates to isolated antibody or antigen-binding portion thereof thatselectively binds to an extracellular domain of a mammalian guanylylcyclase (rGC) receptor in the presence of a ligand or an activatingprotein specific to the mammalian rGC such that the binding of theantibody or antigen binding portion thereof increases the apparentaffinity of the ligand or activating protein for the mammalian rGC. Inspecific embodiments it is shown that the increased affinity of theligand is seen as a slower off-rate of the ligand from the mammalian rGCwhich leads to an apparent increase in affinity. In general terms, themethods described herein result in an increase in molecular potency ofthe ligand making the ligand more effective in its biological responseat lower concentrations. A highly potent drug evokes a larger responseat low concentrations. As potency is a measure of drug's activityexpressed in terms of the amount required to produce an effect of givenintensity, “increased potency” of ligand using the methods of theinvention generally means that less ligand or activating protein isrequired to produce a response in the presence of antibody orantigen-binding portion described herein as compared to the amount ofligand or activating protein that would be required to produce thatlevel of response in the absence of said antibody or antigen-bindingportion thereof that selectively binds to an extracellular domain of amammalian guanylyl cyclase (rGC) receptor.

The mammalian rGC is preferably a human rGC, such as for example, NPRA,NPRB, NPRE or NPRF. In specific embodiments, the human rGC is NPRA.

The ligand in certain defined embodiments is an atrial natriureticpeptide (ANP), a brain natriuretic peptide (BNP), a C-type natriureticpeptide (CNP), a Dendroaspis natriuretic peptide (DNP), urodilatin(Uro), Pseudocerastes persicus natriuretic peptide, snake venomnatriuretic-like peptide A (TNP-a), snake venom natriuretic-like peptideB (TNP-b), or snake venom natriuretic-like peptide C (TNP-c) or activepeptides derived from them or encompassing them.

In specific embodiments, the human rGC is NPRA and the ligand is ANP,BNP, Uro, or a mixture thereof. In other embodiments, the human rGC isNPRA and the ligand is ANP or BNP. In still other embodiments, the humanrGC is NPRB and the ligand is CNP. In yet another embodiments, the humanrGC is GC-C, GC-D, GC-E, GC-F or GC-G (Kuhn, Circ Res 93:700, 2003).

In specific aspects the increase in apparent affinity of the ligand orthe activating protein to the mammalian rGC is at least 2-fold higherthan it would be if the antibody or antigen-binding portion thereof wasnot present. In other embodiments, the EC₅₀ of ligand inducedintracellular cGMP production is at least 2-fold lower than it would beif the antibody or antigen-binding portion thereof was not present.

In certain embodiments, the isolated antibody or antigen-binding portionthereof is such that binding of the antibody or antigen-binding portionthereof enhances or prolongs the catalytic activity associated with themammalian rGC. The catalytic activity is seen to be enhanced orprolonged due to there being an enhanced response of the receptor toligand such that more cGMP is produced at lower ligand concentrations.In specific embodiments, the catalytic activity is an increase inintracellular cGMP production where the rGC is NPRA or NPRB.

In specific embodiments, the antibody or antigen-binding portion thereofis a monoclonal antibody. In more specific embodiments, the antibody isan IgG antibody and more particularly an IgG4 or an IgG1 antibody

In other embodiments, the antibody or antigen-binding portion thereof isselected from the group consisting of: a human antibody, a humanizedantibody, a murine antibody, a chimeric antibody, a peptibody, a singlechain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a Fv fragment, scF_(v) fragment and a fusion protein.

Another aspect of the invention relates to an isolated antibody orantigen-binding portion thereof that selectively binds to an epitopelocated in an extracellular domain of a mammalian receptor guanylylcyclase (rGC) wherein the epitope forms when the rGC is bound to aligand or activating protein specific to the mammalian rGC and whereinthe antibody is not reactive with the ligand or activating proteinbinding site of the rGC. In specific embodiments, the antibodypotentiates the activity of the ligand or activating protein through thereceptor.

In certain embodiments, the antibody or antigen-binding portion thereofselectively binds an epitope located in the extracellular domain ofNPRA, wherein the epitope comprises residues 7-28(NLTVAVVLPLANTSYPWSWARV, SEQ ID NO:30), 121-129 (VKDEYALTT, SEQ IDNO:31), 313-320 (TMEDGLVN, SEQ ID NO:32), 327-333 (HDGLLLY, SEQ IDNO:33) and 347-351 (VTDGE, SEQ ID NO:34) located in the threedimensional structure of NPRA when NPRA is bound to ANP and/or BNP.

In still other embodiments, the antibody or antigen-binding portionthereof selectively binds an epitope located in the extracellular domainof NPRA, wherein the epitope comprises one or more of the sequenceslocated in the three dimensional structure of NPRA when NPRA is bound toANP and/or BNP. Such peptides include the pink portion shown in FIG. 22,which are produced by peptide sequences 28-87(VGPAVELALAQVKARPDLLPGWTVRTVLGSSENALGVCSDTAAPLAAVDLKWEHNPAVF L) (SEQ IDNO:35), 96-113 (APVGRFTAHWRVPLLTAG) (SEQ ID NO:36), 293-301 (PEYLEFLKQ)(SEQ ID NO:37), 310-312 (FNF), 334-335 (IQ), 352-362 (NITQRMWNRSF) (SEQID NO:38).

Certain aspects of the invention relate to an isolated antibody orantigen-binding portion that selectively binds an epitope located in theextracellular domain of NPRA when the NP-A is bound to a ligand, theepitope being defined by a discontinuous region formed by the peptidesshown above for NPRA.

Also contemplated is a monoclonal antibody having the same epitopespecificity as monoclonal antibodies described herein wherein themonoclonal antibody is produced by host cells engineered to expressnucleic acids that encode said antibody molecules.

Also contemplated is a specific isolated antibody or antigen-bindingportion thereof that selectively binds to an epitope located in anextracellular domain of a mammalian receptor guanylyl cyclase (rGC) inthe presence of a ligand or activating protein specific to the mammalianrGC, is not reactive with the ligand or activating protein binding siteof the rGC and competes for binding to said epitope with a monoclonalantibody selected from the group consisting of an antibody comprising aVH and VL chain, each VH and VL chain comprising hypervariable regionsCDR1, CDR2 and CDR3 separated by framework amino acid sequences, thehypervariable regions having amino acid sequences in each VH and VLwherein

VH^(CDR1) of said antibody has a sequence of SEQ ID NO:3;

VH^(CDR2) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:4, 13 and 10,

VH^(CDR3) of said antibody has a sequence of SEQ ID NO: 13,

VL^(CDR1) of said antibody has a sequence of SEQ ID NO: 14,

VL^(CDR2) of said antibody has a sequence of SEQ ID NO: 15; and

VL^(CDR3) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:16, 17 and 18 or an antibody shown in Tables 1and 2.

Another aspect of the invention relates to an isolated antibody orantigen-binding portion thereof that binds to an epitope that isessentially the same epitope bound by an antibody comprising a VH and VLchain, each VH and VL chain comprising hypervariable regions CDR1, CDR2and CDR3 separated by framework amino acid sequences, the hypervariableregions having amino acid sequences in each VH and VL wherein

VH^(CDR1) of said antibody has a sequence of SEQ ID NO:3;

VH^(CDR2) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:4 and 10,

VH^(CDR3) of said antibody has a sequence of SEQ ID NO: 13,

VL^(CDR1) of said antibody has a sequence of SEQ ID NO:14,

VL^(CDR2) of said antibody has a sequence of SEQ ID NO: 15; and

VL^(CDR3) of Said antibody has a sequence selected from the groupconsisting of SEQ ID NO:16, 17 and 18.

Other specific isolated antibody or antigen-binding portion thereofcontemplated are those that bind to an epitope in an extracellulardomain of a mammalian receptor guanylyl cyclase (rGC) in the presence ofa ligand or activating protein specific to the mammalian rGC, whereinsaid epitope is bound by an antibody comprising a heavy chain variableregion (V_(H)) and a light chain variable region (V_(L)), each V_(H) andV_(L) comprising hypervariable regions CDR1, CDR2 and CDR3 separated byframework amino acid sequences, the hypervariable regions having aminoacid sequences in each VH and VL chain of each VH and VL chains of:

VH^(CDR1) having a sequence of SEQ ID NO:3;

VH^(CDR2) having a sequence selected from the group consisting of SEQ IDNO:4 and 10,

VH^(CDR3) having a sequence of SEQ ID NO:13,

VL^(CDR1) having a sequence of SEQ ID NO: 14,

VL^(CDR2) having a sequence of SEQ ID NO:15; and

VL^(CDR3) having a sequence selected from the group consisting of SEQ IDNO:16, 17 and 18.

Other preferred antibodies are those specific for NPRA, comprising: a) aheavy chain variable region having the following amino acid sequencesfor complementarity determining regions, respectively: GDSVSSNSAAWS (SEQID NO:39); RTYYR-SHWYFEYAVSVKS (SEQ ID NO:40) and MDVPSFRYFDV (SEQ IDNO:41) and; b) a light chain variable region having the following aminoacid sequences for complementarity determining regions, respectively:RASQSVRS- - - - -NYLA (SEQ ID NO:42), GASNRAT (SEQ ID NO:43) andQQISNPP-V (SEQ ID NO:44), wherein said antibody binds NPRA in thepresence of an NPRA ligand and increases the affinity of said ligand forsaid NPRA as compared to the affinity of said ligand for said NPRA inthe absence of said antibody.

Also contemplated are cell lines that produce an antibody orantigen-binding portion of an antibody described herein. Specific celllines include those that produce an antibody wherein said antibody has aheavy and light chain CDRs 1, 2 and 3, wherein

VH^(CDR1) of said antibody has a sequence of SEQ ID NO:3;

VH^(CDR2) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:4 and 10,

VH^(CDR3) of said antibody has a sequence of SEQ ID NO:13,

VL^(CDR1) of said antibody has a sequence of SEQ ID NO: 14,

VL^(CDR2) of said antibody has a sequence of SEQ ID NO:15; and

VL^(CDR3) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:16, 17 and 18.

Also contemplated is an isolated nucleic acid molecule comprising asequence encoding an antibody or antigen-binding portion thereof whichbinds to natriuretic peptide receptor A (NPR-A), wherein said antibodyor antigen-binding portion thereof comprises a heavy chain hypervariableregion CDR2 having a sequence selected from the group consisting of SEQID NO:4 and 10.

Other embodiments describe an isolated nucleic acid molecule comprisinga sequence encoding an antibody or antigen-binding portion thereof whichbinds to natriuretic peptide receptor A (NPRA), wherein said antibody orantigen-binding portion thereof comprises a light chain hypervariableregion CDR3 having a sequence selected from the group consisting of SEQID NO:16, 17 and 18.

Yet another embodiment relates to an isolated nucleic acid moleculecomprising a sequence encoding an antibody or antigen-binding portionthereof which binds to natriuretic peptide receptor A (NPRA), whereinsaid antibody or antigen-binding portion thereof comprises a heavy chainhypervariable region CDR2 having a sequence selected from the groupconsisting of SEQ ID NO:4 and 10 and a light chain hypervariable regionCDR3 having a sequence selected from the group consisting of SEQ IDNO:16, 17 and 18.

Another aspect of the invention describes a nucleic acid moleculecomprising a sequence encoding an antibody or antigen-binding portionthereof which binds to natriuretic peptide receptor A (NPRA), whereinsaid antibody or antigen-binding portion thereof comprises a heavy chainvariable domain (V_(H)), wherein the V_(H) comprises hypervariableregions VH^(CDR1) having a sequence of SEQ ID NO:3; VH^(CDR2) having asequence selected from the group consisting of SEQ ID NO:4 and 10, andVH^(CDR3) of said antibody has a sequence of SEQ ID NO: 13, wherein thehypervariable regions are separated by framework amino acid sequences.Also contemplated are isolated nucleic acid molecules comprising asequence encoding an antibody or antigen-binding portion thereof whichbinds to natriuretic peptide receptor A (NPRA), wherein said antibody orantigen-binding portion thereof comprises a light chain variable domain(V_(L)), wherein the V_(L) comprises hypervariable regions VL^(CDR1)having a sequence of SEQ ID NO: 14; VL^(CDR2) having a sequence of SEQID NO:15; and VL^(CDR3) having a sequence selected from the groupconsisting of SEQ ID NO: 16, 17 and 18, wherein the hypervariableregions are separated by framework amino acid sequences. In specificembodiments, the hypervariable regions are provided in a human frameworkregion.

The present invention also relates to isolated nucleic acid moleculecomprising a sequence encoding an antibody or antigen-binding portionthereof which binds to natriuretic peptide receptor A (NPRA), whereinsaid antibody or antigen-binding portion thereof is selected from thegroup consisting of: Ab1, Ab2, Ab3, Ab4 and Ab5, wherein Ab1-Ab5comprises a VH and VL chain, each VH and VL chain comprisinghypervariable regions CDR1, CDR2 and CDR3 separated by framework aminoacid sequences, the hypervariable regions having amino acid sequences ineach VH and VL chain of Ab1-Ab5 selected according to the followingtable:

Ab1: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ IDNO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) Ab2: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQID NO: 4) (SEQ ID NO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 17) Ab3: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3)(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 18) Ab4: VH^(CDR1) VH^(CDR2)VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13) VL^(CDR1)VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 17) Ab5:VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO:13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQID NO: 18).Also contemplated are vectors comprising and capable of expressing thenucleic acid molecule encoding an antibody or antigen-binding portiondescribed herein and host cells transformed with such vectors. Such hostcells may be bacterial host cells or mammalian host cells. Alsocontemplated is a method of producing an isolated antibody orantigen-binding portion thereof comprising the steps of culturing such ahost cell. In specific embodiments, the nucleic acid molecule encodingthe heavy and light chain hypervariable regions separated by frameworkamino acid sequences is coexpressed in the host cell. under suitableconditions and recovering the antibody or antigen-binding portionthereof.

Also contemplated is a pharmaceutical composition comprising thepurified antibody or antigen-binding portion thereof according to anyone of claims 1-36 and a pharmaceutically acceptable carrier orexcipient thereof.

The pharmaceutical composition of claim 50, wherein the antibody ismonoclonal anti-natriuretic peptide receptor A antibody comprises a VHand VL chain, each VH and VL chain comprising hypervariable regionsCDR1, CDR2 and CDR3 separated by framework amino acid sequences, thehypervariable regions having amino acid sequences in each VH and VLchain of Ab1-Ab5 selected according to the following table:

Ab1 : VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQID NO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) Ab2: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQID NO: 4) (SEQ ID NO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 17) Ab3: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 13) VL^(CDR1) VL^(CDR2) VL^(CDR3)(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 18) Ab4: VH^(CDR1) VH^(CDR2)VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13) VL^(CDR1)VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 17) Ab5:VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO:13) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQID NO: 18).

Also contemplated is a method for increasing the apparent affinity of aligand or an activating protein to an extracellular domain of amammalian receptor guanylyl cyclase (rGC), comprising contacting themammalian rGC with the purified antibody or antigen-binding portionthereof according to any one of claims 1-36 in the presence of theligand or activating protein. The potentiation may be performed in vivoor in vitro.

Also contemplated herein are methods of treating a disorder or conditionassociated with a decreased level of catalytic activity of a mammalianrGC in a subject comprising administering to the subject in need thereofan effective amount of a purified antibody or antigen-binding portiondescribed herein.

Other treatment methods include methods of treating a disorder orcondition associated with a decreased level of catalytic activity of amammalian rGC in a subject comprising administering to the subject inneed thereof an effective amount of the pharmaceutical compositiondescribed herein.

The disorders to be treated include but are not limited to a disorderselected from the group consisting of heart failure, hypertension,resistant hypertension, pulmonary hypertension, atherosclerosis,diabetes, diabetic nephropathy, stroke, atrial fibrillation, ventriculararrythmias, deep vein thrombosis, myocarditis, valvular heart disease,pulmonary embolism, pericardial disease, coronary vasopasm, metabolicsyndrome X, renal insufficiency (CKD, ESRD), obesity, asthma andallergic rhinitis.

The heart disease may be selected from the group consisting ofnon-ischemic chronic heart failure, post myocardial infarction heartfailure (ischemic CHF), acute myocardial infarction, reperfusion injury,left ventricular dysfunction, cardiac fibrosis, diastolic heart failure,hypertrophic cardiomyopathy, acute decompensated heart failure andischemic heart disease.

In specific embodiments, the invention involves treating acutedecompensated heart failure in a subject comprising administering to thesubject in need thereof an effective amount of the pharmaceuticalcomposition according to the invention.

Other methods involve treating hypertension resistant hypertension orpulmonary hypertension in a subject comprising administering to thesubject in need thereof an effective amount of a purified antibody orantigen-binding portion or a pharmaceutical composition comprising suchan antibody or antigen binding portion thereof.

Other methods involve treating atherosclerosis in a subject comprisingadministering to the subject in need thereof an effective amount of apurified antibody or antigen-binding portion or a pharmaceuticalcomposition comprising such an antibody or antigen binding portionthereof.

Other methods involve treating a disease in a subject, the disease beingselected from the group consisting of Type 1 diabetes. Type 2 diabetes,diabetic nephropathy, stroke, atrial fibrillation/ventriculararrhythmias, deep vein thrombosis, myocarditis, valvular heart disease,pulmonary embolism, pericardial diseases, coronary vasospasm, metabolicsyndrome X, renal insufficiency, allergic rhinitis, asthma, inflammatorydisease, septic shock, obesity and cancer, comprising to the subject inneed thereof an effective amount of a purified antibody orantigen-binding portion thereof described herein or a pharmaceuticalcomposition comprising such an antibody or antigen binding portionthereof.

Also contemplated are methods of treating heart failure in a patientcomprising administering a therapeutically effective amount of apurified antibody or antigen-binding portion thereof according topresent invention and a second therapeutic agent for the treatment ofheart failure. For example, the second agent may be selected from thegroup consisting of ARB/ACEi, ADP inhibitors, aldosterone antagonists,natieuretic peptides, anti-arrhythmic agents, HMG-CoA inhibitors, betablockers, cardiac glycosides, calcium channel blockers, diuretics,fibrates, GPIIb/IIIa inhibitors, heparins, nicotinic acid derivatives,nitrates and nitrites, oral anticoagulants, thrombolytics, TZDs,cholesterol absorption inhibitors, acetyl salicylic acid, diapyridamole,phosphodiesterase inhibitors, CETP inhibitors/apoA1 mimetics, thrombininhibitors, Factor Xa inhibitors, renin inhibitors, chymase inhibitors,RhoK inhibitors, LpPLA2 inhibitors, Endothelin receptor antagonists,HDAC inhibitors, nuclear receptor agonists, nuclear receptorantagonists, vasopeptidase inhibitors, fatty acid oxidation inhibitors,ACAT inhibitors, microsomal triglyceride transfer protein inhibitors,adenosine receptor modulators, AGE/RAGE interaction modulators, genetherapy, cell therapy.

In specific embodiments, the second agent is ANP, BNP or urodilatin.More specifically, the second agent is selected from the groupconsisting of Nesiritide, Carperitide, Ularitide, and combinationsthereof.

Other methods of the invention comprise prolonging or increasing thetherapeutic efficacy of an NPRA ligand in a patient comprisingadministering the NPRA ligand in combination with an antibody describedherein or antigen binding portion of such an antibody. In specificaspects, the patient is suffering from heart failure. More specifically,the patient is suffering from acute decompensated heart failure. Inother aspects, the patient is suffering from chronic heart failure. TheNPRA ligand may be selected from the group consisting of is selectedfrom the group consisting of Nesiritide, Carperitide, Ularitide, andcombinations thereof. In specific aspects, co-administering the antibodywith the NPRA ligand prolongs the effect of the NPRA ligand by at leasttwo-fold the time of the NPRA ligand activity seen in the absence of theantibody. In specific embodiments, the antibody is administeredconcurrently with, prior to, or after administration of the NPRA ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B: Binding of 5064 to HEK NPRA cells (FIG. 1A) andnon-transfected HEK cells (FIG. 1B) in presence or absence of ANP orBNP. 5064 Fab. 5064 Fab-dHLX, a negative control Fab-dHLX (n.c.dHLX) and4880 Fab are tested at 20, 2 and 0.2 μg/ml for binding to HEK NPRA cellsand untransfected cells by FACS. 4880 Fab serves as a positive controlas it is a Fab that binds to NPRA in the absence or presence of ANP orBNP. MFI—mean fluorescent intensity

FIG. 2A, FIG. 2B and FIG. 2C: Dose Response of Binding of 5064 inDifferent Formats to NPRA transfected HEK cells. FIG. 2A and FIG. 2B:Binding of 5064 in Fab and Fab-dHLX formats +/−ANP or BNP to HEK NPRA isdetermined by FACS. An irrelevant Fab or Fab-dHLX are utilized as anegative controls. FIG. 2C: Binding of 5064 in IgG format is compared to5064 Fab binding by FACS.

FIG. 3: Demonstration of Binding of 5064 Fab-dHLX to NPRA-Fc by ELISA.Binding was tested in presence and absence of ligands. Binding tocontrol Fc antigen (neg control antigen) was tested in presence andabsence of ANP.

FIG. 4: 5064 Fab-dHLX Does Not Bind to Biotinylated ANP Alone by ELISA.5064 Fab-dHLX, a negative control Fab-dHLX, and an anti-ANP mousemonoclonal antibody are tested for binding to plate bound biotinylatedANP by ELISA.

FIG. 5: Free ANP Does Not Compete with ANP Complexed NPRA for Binding of5064 Fab. 5064 Fab is pre-incubated with various concentrations of ANPprior to adding to HEK NPRA cells loaded with 100 nM ANP. Binding wasanalyzed in FACS. MFI—mean fluorescent intensity

FIG. 6: 5064-dHLX Enhances NPRA-mediated cGMP Production in the Presenceof Suboptimal Concentrations of ANP. HEK NPRA cells are incubated in thepresence or absence of increasing concentrations of 5064-dHLX or acontrol antibody and 40 pM ANP and cGMP is measured as described.

FIG. 7: 5064 Enhances NPRA-mediated cGMP Production in the Presence ofSuboptimal Concentrations of BNP. HEK NPRA cells are incubated in thepresence or absence of increasing concentrations of 5064-Fab dHLX orIgG, or control (3207) and 40 nM BNP and cGMP is measured as described.

FIG. 8A and FIG. 8B: Effects of 5064 Fab and Fab-dHLX on ANP and BNPstimulated cGMP responses in HEK NPRA cells. 20 μg/ml 5064 Fab orFab-dHLX or control Fabs are incubated with HEK NPRA cells in thepresence of increasing concentrations of ANP (FIG. 8A) or BNP (FIG. 8B)and cGMP is quantitated as described.

FIG. 9: The 5064 IgG uniquely potentiates ANP mediated NPRA activation.200 nM 5064 IgG1, and other NPRA binders 4878, 4879, 4880 are added toNPRA HEK cells in the presence of increasing concentrations of ANP andcGMP is measured as described.

FIG. 10: Affinity matured Fabs bind to HEK NPRA cells complexed with ANPto a greater extent than the parental Fab 5064. HEK NPRA cells areloaded with 100 nM ANP. Whole cell Fab binding is monitored by FACSanalysis and the non-potentiating NPRA-specific Fab 4880 and negativecontrol Fab 3207 are also included as controls. MFI—mean fluorescentintensity

FIG. 11: Affinity matured Fabs enhance the cGMP response in HEK NPRAcells to suboptimal levels of ANP. HEK NPRA cells are incubated with 400nM Fabs and 400 pM ANP. cGMP is quantitated as described.

FIG. 12A and FIG. 12B: Affinity matured Fabs enhance the NPRA mediatedcGMP response to both ANP and BNP. HEK NPRA cells are incubated with 400nM Fabs and increasing concentrations of ANP (FIG. 12A) or BNP (FIG.12B). cGMP is quantitated as described.

FIG. 13A and FIG. 13B: Affinity matured (FIG. 13A) and cross combined(FIG. 13B) IgG4_Pro antibodies bind to ANP loaded NPRA with betteraffinity than the parental antibody 5064. HEK NPRA cells are incubatedwith 100 nM ANP and increasing concentrations of antibodies and bindingis determined by FACS. MFI—mean fluorescent intensity

FIG. 14A and FIG. 14B: Affinity matured (FIG. 14A) and cross combined(FIG. 14B) IgG4_Pro antibodies enhance the NPRA mediated cGMP responseto ANP. HEK NPRA cells are incubated with 200 nM antibody and increasingconcentrations of ANP. cGMP is quantitated as described. 5064 is theparental antibody and 3207 is a control antibody.

FIGS. 15A and 15B: Affinity matured (FIG. 15A) and cross combined (FIG.15B) IgG4_Pro antibodies enhance the NPRA mediated cGMP response to BNP.HEK NPRA cells are incubated with 200 nM antibody and increasingconcentrations of BNP. cGMP is quantitated as described. 5064 is theparental antibody and 3207 is a control antibody.

FIG. 16A and FIG. 16B: Affinity matured (FIG. 16A) and cross combined(FIG. 16B) IgG_Pro antibodies do not cross react with NPRB. HEK NPRAcells +/−100 nM ANP, HEK NPRB cells +/−100 nM CNP and non-transfectedHEK cells +/−100 nM ANP or CNP are incubated with 20 nM of antibody andbinding is assessed by FACS. 3207 is included as a negative controlantibody. MFI—mean fluorescent intensity

FIG. 17: Affinity matured and cross combined IgG_Pro antibodies do notcross react with NPRC. HEK NPRA cells +/−100 nM ANP or BNP or HEK NPRCcells +/−100 nM ANP or BNP are incubated with 20 nM of antibody andbinding is assessed by FACS. 5064 is the parental antibody.

FIG. 18. KinExA binding curve used for calculating the apparent K_(d) of5591 IgG binding to HEK NPRA cells. 75 pM (black, square) and 200 pM(red, circle) 5591 IgG

FIG. 19. 5591 Potentiates NPRA-mediated cGMP Responses over time in HEKNPRA Cells. HEK NPRA cells are incubated with 0, 200 pM or 1 μM ANP inthe absence or presence of 10 μg/ml 5591 IgG and cGMP levels arequantitated up to 120 minutes.

FIG. 20. 5502, 5504 and 5064 enhance the binding of ¹²⁵I ANP to HEK NPRAcells as compared to the control antibody 3207. HEK NPRA cells areincubated with increasing concentrations of ¹²⁵I ANP in the presence ofanti-NPRA or control antibodies and cell associated radioactivity isquantitated. Non-specific binding in determined by adding an excess ofcold ANP. CPM/well=counts per minute/well

FIG. 21. 5502, 5504, 5591, 5592 and 5064 slow the off-rate of ¹²⁵I ANPfrom HEK NPRA cells. HEK NPRA cells are incubated in the presence ofanti-NPRA or control antibodies and 100 nM ¹²⁵I ANP. Excess cold ANP isthen added and the cells associated radioactivity is quantitated overtime. The points to the left of the zero timepoint reflect cellassociated radioactivity prior to the addition of cold ANP. CPM=countsper minute

FIG. 22: Mapping of peptides from the extracellular domain of NPRAshowing changes in deuterium exchange rates upon 5591 binding. Areasshaded in pink represent strong protection from exchange and areasshaded in red very strong protection. The areas shaded in red arerepresented by an epitope that comprises residues 7-28(NLTVAVVLPLANTSYPWSWARV) (SEQ ID NO:30), 121-129 (VKDEYALTT) (SEQ IDNO:31), 313-320 (TMEDGLVN) (SEQ ID NO:32), 327-333 (HDGLLLY) (SEQ IDNO:33) and 347-351 (VTDGE) (SEQ ID NO:34) located in the threedimensional structure of NPRA when NPRA is bound to ANP and/or BNP andthe areas shaded in pink represent epitopes that have peptide sequences28-87 (VGPAVELALAQVKARPDLLPGWTVRTVLGSSENALGVCSDTAAPLAAVDLKWEHNPAVF L)(SEQ ID NO:35), 96-113 (APVGRFTAHWRVPLLTAG) (SEQ ID NO:36), 293-301(PEYLEFLKQ) (SEQ ID NO:37), 310-312 (FNF), 334-335 (IQ), 352-362(NITQRMWNRSF) (SEQ ID NO:38).

DETAILED DESCRIPTION OF THE INVENTION

Heart failure remains a common and growing public health concern in theindustrialized world. With currently available therapies approximately50% of patients with heart failure die within 5 years of theirdiagnosis. The inadequacies of the currently approved therapies forheart failure have led to a continued search for robust and efficienttherapeutic compositions that can be used in the treatment of acutedecompensated heart failure as well as management of chronic heartfailure. The present disclosure provides methods and compositions forpotentially meeting this unmet need.

In general, the invention provides novel antibody compositions andmethods of using therapeutically effective amounts of the same, eitheralone or in combination with other therapeutic agents, for the treatmentor prevention of disease. The term “guanylyl cyclase or GC” refers to afamily of enzymes (lyases) that catalyze the conversion of guanosinetriphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP) andpyrophosphate. GCs are subdivided into two forms: (1) soluble GCs and(2) particulate GCs. The soluble GCs can be activated by nitric oxide,whereas particulate GCs can be activated by peptide hormones, includingnatriuretic peptides. Particulate GCs are also referred to as receptorguanylyl cyclases (rGCs).

The term “natriuretic peptide receptors” as used herein refers tomembrane-bound receptors that are bound by natriuretic peptides.Guanylyl cyclase A (GC-A isoform) or natriuretic peptide receptor A(NPRA) acts as the receptor for the natriuretic peptides ANP, urodilatinand BNP. The sequence of human NPRA is known to those of skill in theart. For example, a human NPRA nucleic acid sequence has been depositedat Genbank accession No. NM_000906. The nucleic acid sequence for NPRAis reproduced herein as SEQ ID NO: 1. The amino acid sequence of NPRA isreproduced herein as SEQ ID NO:2. Guanylyl cyclase B (GC-B) ornatriuretic peptide receptor B (NPRB) serves as the receptor for thenatriuretic peptide CNP. Additionally, natriuretic peptide receptor C(NPRC) acts as a receptor for ANP, urodilatin, BNP and CNP. The clonesof these various receptors may readily be purchased from OriGeneTechnologies, Inc. (Rockville, Md.).

The term “natriuretic peptides or ligands” refers to a family of peptidehormones each containing a 17-amino acid long ring that is closed by adisulfide bond between two cysteine residues. A “ligand” is any moleculethat binds to another molecule via non-covalent bonds. In biologicalprocesses, a ligand, binds specifically to another molecule, such as anenzyme or protein receptor, and is either transformed into somethingelse or initiates a cellular process. ANP, urodilatin, BNP and CNPrepresent peptide hormones ligands that bind to pGCs. As mentionedabove. ANP, urodilatin, and BNP bind to and activate NPRA. As usedherein, the term “natriuretic peptide receptor A (NPRA) or guanylatecyclase A (GC-A)” may be used interchangeably with the following termsand/or acronyms: atrionatriuretic peptide receptor A, ANPa, ANP-A,ANPRA, Atrial natriuretic peptide A-type receptor, Atrial natriureticpeptide receptor A precursor, GUC2A, GUCY2A and NPR-A.

The biological activities associated with NPRA activation include, butare not limited to, vasodilation, diuresis, natriuresis, inhibition ofcardiac remodeling, anti-fibrotic effects, anti-inflammatory effects,lipolysis and decreased sympathetic nervous system activity (see, e.g.,Levin et al., NEJM, 339:321, 1998; Kuhn, Circulation Res., 93:700, 2003;and Denus et al., Chest, 125:652, 2004). The compositions of theinvention may be used in the treatment of a variety of diseases thatinvolve these cGMP-mediated biological activities. Such diseases includebut are not limited to disorders such as hypertension, resistanthypertension, pulmonary hypertension, chronic heart failure, acutedecompensated heart failure, myocardial infraction, stable, unstable andvariant (Prinzmetal) angina, atherosclerosis, cardiac edema, renalinsufficiency, nephrotic edema, hepatic edema, stroke, asthma,bronchitis, chronic obstructive pulmonary disease (COPD), cysticfibrosis, dementia, immunodeficiency, premature labor, dysmenorrhoea,benign prostatic hyperplasis (BPH), bladder outlet obstruction,incontinence, conditions of reduced blood vessel patency, e.g.,postpercutaneous transluminal coronary angioplasty (post-PTCA),peripheral vascular disease, allergic rhinitis, cystic fibrosis, andglucoma, and diseases characterized by disorders of gut motility, e.g.,irritable bowel syndrome (IBS). In these methods, the antibodycompositions and/or the additional therapeutic agents can beadministered separately or as components of the same composition in oneor more pharmaceutically acceptable carriers.

Particular aspects described herein relate to the treatment of variousheart conditions, including chronic heart failure, hypertension,unstable angina, sudden cardiac death, and acute myocardial infarction,and particularly, acute decompensated heart failure. As describedherein, natiuretic peptides such as ANP, and BNP are elevated inbiological samples from patients with failing hearts but are at lowlevels in biological samples from control patients. BNP and ANP areligands for the receptor NPRA. The antibodies of the present inventionspecifically recognize the activated ligand-receptor complex. Indeed,studies presented herein show little or no binding by the antibodies toeither the receptor alone or to either of the ligands alone. Thus theantibodies of the invention are powerful and specific potentiators ofthe ligands only in the activated receptor complex. As these antibodiespotentiate the effects of the activated complex (e.g., by prolonging theeffect or by increasing the magnitude of the effect), these antibodiespresent a significant advance over the currently available recombinantpeptide based therapies which are short lived.

Thus, it is contemplated that the antibody compositions described hereincan be used in the therapeutic intervention of any disorder in which itis desired to increase, potentiate, or otherwise upregulate theproduction of cGMP mediated through the activated NPRA-ligand complex.While the antibodies alone have been shown to be effective inpotentiating the effects of the NPRA receptor, it is contemplated thatthe antibody compositions also will be useful when used in combinationwith existing therapeutic compositions for the treatment of heartfailure. In particular, it is contemplated that the antibodycompositions will be used in combination with recombinant natiureticpeptides or derivatives thereof. Intravenous therapy with recombinantBNP (Nesiritide, Natrecor®.) significantly decreases pulmonary capillarywedge pressure and systemic vascular resistance and increases cardiacindex. BNP is not pro-arrhythmic and has no effect on heart rate. Burgerand Burger, Curr. Opin. Investig. Drugs 2:929 2001. It is contemplatedthat the antibody compositions will be administered in combination withNesiritide. It is contemplated that such a combination therapyadministration will result in decreased pulmonary capillary wedgepressure and systemic vascular resistance and increased cardiac index inan amount and manner that the therapeutic effect is longer than theeffect typically observed in the use of Nesiritide alone. Thus, oneembodiment described herein is a method of increasing the time and/ormagnitude of one or more of decreases of pulmonary capillary wedgepressure and systemic vascular resistance and increased cardiac indexobserved on the treatment with recombinant natuiretic peptide.

Thus in some embodiments, the antibody compositions provided herein canbe used to stimulate cGMP and/or vasodilate arteries in a mammal. Inaddition, the antibody compositions provided herein can be used to treathypertension, pulmonary hypertension, resistant hypertension, acutedecompensated heart failure and/or chronic heart failure. In otherembodiments, the antibodies particularly are useful in increasingdiuresis and/or natriuresis in a mammal. For example, an antibodycomposition described herein can be administered to a mammal to increaseurinary flow and urinary excretion of sodium. In addition, theantibodies can be used to treat a fluid overload state (e.g., chronicheart failure, liver failure, and kidney failure) and/or to treat asodium overloaded state (e.g., chronic heart failure and kidneyfailure).

Antibody Compositions

The present invention relates to antibody compositions that specificallybind the activated NPRA-ligand complex and potentiate the effects of theligand of that receptor.

Typically, the term “agonist” refers to a ligand that binds to areceptor and activates it biological activity. “Agonistic activity” isdefined as activation of a pGC leading to the production of cGMP. Insome narrower and specific aspects, the term “NPRA agonist” is used torefer to an agent that causes an activation of an NPRA in the absence ofits ligands ANP or BNP.

The term “potentiator” refers to a molecule(s) that affects the“on-rate” or “off-rate” of a ligand binding to its receptor and causesan increase in the effectiveness and/or duration of the agonisticactivity. “Potentiating activity” is defined as an enhanced activationof a pGC in the presence of suboptimal concentrations of its ligands.

The following table provides exemplary CDR 1, CDR 2, CDR3 regions of theheavy and light chains of preferred antibodies of the present invention.

TABLE 1 HEAVY CHAIN Antibody VH CDR1 VH CDR2 VH CDR3 5064 GDSVSSRSASWSRIYYRSKWYNDIAVSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 13) 5503 GDSVSSRSASWS RTYIRSHWIFEYAGSVNIS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 5) (SEQ ID NO: 13) 5515 GDSVSSRSASWSRTYYRSHWYWEYADSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 6)(SEQ ID NO: 13) 5505 GDSVSSRSASWS RTYYRSHWYYEYARSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 7) (SEQ ID NO: 13) 5508 GDSVSSRSASWSRTYYRSHWYFEYAHSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 8)(SEQ ID NO: 13) 5509 GDSVSSRSASWS RTYYRSHWYFDYAVSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 8) (SEQ ID NO: 13) 5510 GDSVSSRSASWSRTYYRSHWYYEYAASVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 9)(SEQ ID NO: 13) 5511 GDSVSSRSASWS RTYYRSHWYYEYAQSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13) 5512 GDSVSSRSASWSRTYYRSHWYMEYAHSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 11)(SEQ ID NO: 13) 5516 GDSVSSRSASWS RTYYRSKWYYEYAHEVKS MDVPSFRYFDV(SEQ ID N): 3) (SEQ ID NO: 12) (SEQ ID NO: 13) 5502 GDSVSSRSASWSRIYYRSKWYNDYAVSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 13) 5504 GDSVSSRSASWS RTYYRSKWYNDYAVSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 4)  (SEQ ID NO: 13) 5507 GDSVSSRSASWSRIYYRSKWYNDYAVSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 13) 5513 GDSVSSRSASWS RIYYRSKWYNDYAVSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 13) 5514 GDSVSSRSASWSRIYYRSKWYNDYAVSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 13)  5591 GDSVSSRSASWS RTYYRSHWYYEYAQSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13) 5592 GDSVSSRSASWSRTYYRSHWYYEYAQSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 10)(SEQ ID NO: 13) 5593 GDSVSSRSASWS RTYYRSHWYYEYAQSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13) 5594 GDSVSSRSASWSRTYYRSHWYYEYAQSVKS MDVPSFRYFDV (SEQ ID NO: 3) (SEQ ID NO: 10)(SEQ ID NO: 13) 5594 GDSVSSRSASWS RTYYRSHWYYEYAQSVKS MDVPSFRYFDV(SEQ ID NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 13)

TABLE 2 LIGHT CHAIN VLI, CDR 1 VL CDR 2 VL CDR 3 5064 RASQSVRSNYLAGASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 5503RASQSVRSNVLA GASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) 5515 RASQSVRSNYLA GASNRAT QQISNPPVT (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 16) 5505 RASQSVPSNYLA GASNRAT QQISNPPVT(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 5508 RASQSVRSNYLAGASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 5509RASQSVRSNYLA GASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) 5510 RASQSVRSNYLA GASNRAT QQISNPPVT (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 16) 5511 RASQSVRSNYLA GASNRAT QQISNPPVT(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 5512 RASQSVRSNYLAGASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 5516RASQSVRSNVLA GASNRAT QQISNPPVT (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) 5502 RASQSVRSNYLA GASNRAT QQISNSPPT (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 17) 5504 RASQSVRSNYLA GASNRAT QQISRAPAT(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 18) 5507 RASQSVRSNYLAGASNRAT QOISTNPPT (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 19) 5513RASQSVRSNYLA GASNRAT QQISSSPAT (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 20) 5514 RASQSVPSNYLA GASNRAT QQISISPPT (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 21) 5591 RAWSVRSNYLA GASNRAT QQISNSPPT(SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 17) 5592 RAWSVRSNYLA GASNRATQQISRAPAT (SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 18) 5593RASQSVRSNYLA GASNRAT QQISTNPPT (SEQ ID NO: 14) (SEQ ID NO: 16)(SEQ ID NO: 19) 5594 RAWSVRSNYLA GASNRAT QQISSSPAT (SEQ ID NO: 14)(SEQ ID NO: 16) (SEQ ID NO: 20) 5595 RAWSVRSNYLA GASNRAT QQISISPAT(SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 21)The framework of regions of the heavy chains for the exemplaryantibodies were as follows:

FR 1 for the VH region:  (SEQ ID NO: 22) QVQLQQSGPGLVKPSQTLSLTCAISFR 2 for the VH region:  (SEQ ID NO: 23) WIRQSPGRGLEWLG FR 3 for the VH region:  (SEQ ID NO: 24)RITINPDTSKNQFSLQLNSVTPEDTAVYYCAR  FR 4 for the VH region: (SEQ ID NO: 25) WGQGTLVTVES The framework of regions of the light chains for the exemplaryantibodies were as follows:

FR 1 for the VL region:  (SEQ ID NO: 26) DIVLTOSPATLSLSPGERATLSCFR 2 thr the VL region:  (SEQ ID NO: 27) WYQQKPGQAPRLLIYFR 3 for the VL region:  (SEQ ID NO: 28)GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC FR 4 for the VL region: (SEQ ID NO: 29) FGQGTKVEIKRT

Given that the present invention has identified unique antibodies thathave therapeutic applicability, each of the heavy and light chainsdepicted in the present application can now be prepared usingrecombinant methods and processed in a recombinant cell line to a matureform. Accordingly, by using recombinant production in mammalian cells,the mature form of the antibody is processed proteolytically and alsoincludes other post translational modifications such as glycosylation.

Nucleic acids encoding light chain variable regions can be constructedand co-expressed with nucleic acids encoding a heavy chain and viceversa, and optionally may be linked to constant regions. Any heavy chainand light chains may be combined as long as suitable NPRA bindingaffinity is maintained. The desired genes encoding the light and heavychains are introduced into mammalian cells and the resultant recombinantimmunoglobulin products are expressed, purified and characterized usingstandard recombinant methods.

An “antibody” generally refers to a protein that recognizes and binds toa specific antigen and an “immunoglobulin” generally refers to aglycoprotein that functions as an antibody. In native form, animmunoglobulin molecule consists of four chains, two identical heavychains (about 50-70 kDa each) and two identical light chains (about 25kDa each), which are held together by disulfide bonds. Specifically,each heavy chain is linked to a light chain by one disulfide bond,whereas the number of disulfide bonds between heavy chains variesdepending on the immunoglobulin isotype (IgG, IgA, IgM, IgD and IgE).Additionally, each heavy chain and each light chain has regularly spacedintrachain disulfide bonds or bridges. In both heavy and light chains,there are constant domains and variable domains. For example, each heavychain (γ, δ, α, μ or ε) has at one end a variable domain (V_(H))followed by a number of constant domains (C_(H1), C_(H2), C_(H3),C_(H4)), whereas each light chain (either λ or κ) has a variable domain(V_(L)) at one end and constant domain (C_(L)) at its other end. C_(L)is aligned with C_(H1) and C_(L) is aligned with V_(L).

It is contemplated that the antibodies of the invention may be an IgGmolecule of any isotype (i.e., the framework may be an IgG1, IgG2, IgG3or an IgG4 type IgG). Depending on the amino acid sequence of theconstant domain of their heavy chains, human immunoglobulins can beassigned to different classes. There are five major classes, IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma and murespectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have ADCC activity.

In certain embodiments, the therapeutic antibodies of the invention areIgG1 molecules. It is now established that IgG4 does not activatecomplement, as such, the chance of an immunogenic response andinflammation due to antigen-antibody-complement complexes is greatlyreduced with the use of IgG4 molecules as compared to other isotypes.This makes IgG4 a very attractive candidate for therapy as it isexpected to be a safe therapeutic modality: IgG4 should simply bind toantigen and should not trigger any additional response in human body.For example, in nature, an IgG4-based response is generated in responseto, for example, antigens such as dust mite, grass pollen or bee sting.These antigens are typically eliminated without significant immuneresponse and inflammation. Thus, in some embodiments it is desirablethat the antibodies of the invention are IgG4 antibodies. Regardless ofthe isotype used, when the compositions are formulated, it is desirableto include in the formulation agents that will allow the conformationand refolding of the isotypes to be as homogeneous as possible by forexample including agents such as chaotropic and redox reagents to limitthe refolding of double bonds (see WO 2006/047340).

As used herein, the term “antibody” refers to an intact immunoglobulinand “an antigen-binding portion thereof” refers to a protein moleculethat competes with the intact antibody for specific binding. An antibodymay be monoclonal, chimeric, humanized, human, CDR-grafted or murineantibody. In specific embodiments, the antibodies described herein arefully human antibodies that are identified through phage display fromhuman combinatorial libraries such as for example HuCal® (Morphosys,Munich, Germany). Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, but are not limitedto, Fab, Fab′, F(ab′)₂, Fv, a single domain antibody (dAb), andcomplementarity determining region (CDR) fragments, single-chainantibodies (scFv), diabodies and polypeptides or fusion proteins thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide such that thepolypeptide still binds to an NPRA receptor and preferably exhibits abiological activity of the antibodies described herein.

An “epitope or determinant” generally relates to a specific chemicaldomain on an antigen that stimulates the production of, and isrecognized by, an antibody. An individual epitope on a molecule such asprotein elicits the synthesis of a different antibody (also known as anantigenic determinant). The epitope is defined by the three dimensionalstructure of the molecule to which the antibody binds. In the presentdisclosure, antibodies have been identified which specifically bind toone monomer of the NPRA dimer that is formed upon binding of ANP to thereceptor. FIG. 29 summarizes mass spectrometry data of the threedimensional structure and epitopes of the NPRA to which the antibodiesof the invention bind.

Hydrogen/Deuterium Mass Spectrometry (HXMS) analysis showed that thestrongest binding is to a region of the extracellular domain of NPRAthat contains the peptides encompassing residues 7-28(NLTVAVVLPLANTSYPWSWARV) (SEQ ID NO:30), 121-129 (VKDEYALTT) (SEQ IDNO:31), 313-320 (TMEDGLVN) (SEQ ID NO:32), 327-333 (HDGLLLY) (SEQ IDNO:33) and 347-351 (VTDGE) (SEQ ID NO:34). These peptides form onediscontinuous region in the three dimensional structure of NPRA whenNPRA has bound thereto ANP.

The term “antigen” refers broadly to any substance that elicits animmune response.

The complementarity determining regions of the NPRA may be inserted intoa unique combination of the human heavy and light chains of structurallydiffering IgG1 and IgG2 and IgG4. The binding of these antibodies toNPRA and their efficacy as NPRA agonists or potentiators of NPRA ligandscan be readily assessed using assays such as those described in theexamples below.

The term “specific binding agent” includes antibodies as defined aboveand recombinant peptides or other compounds that contain sequencesderived from CDRs having the desired antigen-binding properties.Specifically included in the term are peptides containing amino acidsequences that are at least 80%, 90% or 100% identical to one or moreCDRs of NPRA antibodies described herein, preferably including avariable heavy chain CDR2 having a sequence RIYYRSKWYNDYAVSVKS (SEQ IDNO:4) or RTYYRSHWYYEYAQSVKS (SEQ ID NO:10) and/or a light chain CDR3having a sequence of QQISNPPVT (SEQ ID NO:16) or QQISNSPPT (SEQ IDNO:17) or QQISRAPAT (SEQ ID NO:18).

Other antibody-related molecules also are contemplated. In particular,the antibodies of the invention can form the basis for “peptibodies.”These antibody related molecules which comprise an antibody Fc domain asthe “vehicle” attached to at least one antigen-binding peptide. AntibodyCDR's from the NPRA antibodies, particularly those that include theheavy chain CDR2 and/or the light chain CDR3 described above, may besuitable for incorporation into a peptibody. For a more detaileddescription of peptibody production, see WO 00/24782, published May 4,2000. Peptibodies can be made by linking peptides in tandem (i.e.,sequentially) either directly to each other or separated by linkers.Those peptides that contain cysteine residues may be cross-linked withanother cysteine-containing peptide, either or both of which may belinked to a vehicle. Any peptide having more than one Cys residue mayform an intrapeptide disulfide bond, as well. Antibodies technologiesoften will use derivatization of such peptides using for example,capping of the carboxyl terminus with an amino group, capping cysteinesresidues or substituting amino acid residues by moieties other thanamino acid residues (see, e.g., Bhatnagar et al., J. Med. Chem. 39:3814, 1996, and Cuthbertson et al., J. Med. Chem. 40: 2876, 1997). Inaddition, optimization of the peptides for NPRA binding properties akinto the affinity maturation of the antibodies also can be performed.

The antigen binding portion of the antibodies of the invention also canbe modified with various molecules that can be inserted within thepeptide portion itself or between the peptide and vehicle portions ofthe specific binding agents, while retaining the desired activity ofspecific binding agent. Exemplary such insertions include insertion ofan Fc domain, addition of a polyethylene glycol or other relatedmolecules such as dextran, a fatty acid, a lipid, a cholesterol group, asmall carbohydrate, a peptide, a cyotoxic agent, a chemotherapeuticagent, a detectable moiety as described herein (including fluorescentagents, radiolabels such as radioisotopes), an oligosaccharide,oligonucleotide, a polynucleotide, interference (or other) RNA, enzymes,hormones, or the like. Other molecules suitable for incorporation inthis fashion will be appreciated by those skilled in the art, and areencompassed within the scope of the invention. This includes insertionof, for example, a desired molecule in between two consecutive aminoacids, optionally joined by a suitable linker.

An “isolated” antibody is one that has been identified and separatedfrom a component of the cell that expressed it. Contaminant componentsof the cell are materials that would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody, and most preferably more than 99% by weight. (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated naturally occurring antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a complementarity determiningregion or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)]. Even asingle CDR may recognize and bind antigen, although with a loweraffinity than the entire antigen binding site containing all of theCDRs. It is understood that the CDR of an antibody may includeadditional or fewer sequences outside the specified limits above so longas the antibody retains its ability to bind the target molecule.

“Framework” or “FR” residues are those variable region residues otherthan the hypervariable region residues.

“Antibody fragments” comprise a portion of an intact full lengthantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng., 8:1057, 1995); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment which contains the constant region.The Fab fragment contains all of the variable domain, as well as theconstant domain of the light chain and the first constant domain(C_(H1)) of the heavy chain. The Fc fragment displays carbohydrates andis responsible for many antibody effector functions (such as bindingcomplement and cell receptors), that distinguish one class of antibodyfrom another.

Antibodies that are treated with pepsin yield a F(ab)s fragment that hastwo “Single-chain Fv” or “sFv” antibody fragments comprising the VH andVL domains of antibody, both of which are present in a singlepolypeptide chain. Fab fragments differ from Fab′ fragments by theinclusion of a few additional residues at the carboxy terminus of theheavy chain CH1 domain including one or more cysteines from the antibodyhinge region. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the Fv toform the desired structure for antigen binding. For a review of sFv seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, a single variabledomain (or half of an Fv comprising only three CDRs specific for anantigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161 and 30: Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444, 1993.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies, but can also be produced directly byrecombinant host cells. See, for example. Better et al., Science240:1041, 1988; Skerra et al. Science 240: 1038, 1988; Carter et al.,Bio/Technology 10:163, 1992.

Methods of Identifying the Antibodies

Antibodies of the present invention may now be produced usingrecombinant DNA methodology using one of the antibody expression systemswell known in the art (see, e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory (1988)).

The amino acid sequence of the VH and VL regions of interest have beendescribed herein. The suitable encoding nucleotide sequences can bedesigned according to a universal codon table using techniques known tothose of skill in the art. The nucleic acids are then amplified andcloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used is not critical, so long as it ispossible to determine the sequence of some portion of the immunoglobulinpolypeptide of interest.

As used herein, an “isolated” nucleic acid molecule or “isolated”nucleic acid sequence is a nucleic acid molecule that is either (1)identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid or (2) cloned, amplified, tagged, or otherwisedistinguished from background nucleic acids such that the sequence ofthe nucleic acid of interest can be determined. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. However, an isolated nucleic acid molecule includes a nucleicacid molecule contained in cells that ordinarily express the antibodywhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The sequences encoding immunoglobulins or immunoglobulin polypeptidesspecific for binding to NPRA were identified using phage displaytechnology. Phage display is described in e.g., Dower et al., WO91/17271, McCafferty et al., WO 92/01047, and Caton and Koprowski, Proc.Natl. Acad. Sci. USA, 87:6450, 1990, each of which is incorporatedherein by reference. In preferred aspects, the antibodies of the presentinvention were identified using phage display techniques using a humancombinatorial library (HuCal®; Morphosys, Munich, Germany). This librarycomprises more than ten billion different, fully human antibodies.HuCAL® allows rapid and automated production of high-affinityantibodies. The most important feature of the library is its capabilityof optimizing fully human antibodies to meet predefined specifications.Detailed experimental protocols for the identification of the antibodiesdescribed herein are provided in Examples 3 through 6 below. Briefly,however, the antibodies were identified after several pannings wereperformed with the HuCAL GOLD® library in order to select agents fromthe library that had a binding affinity for NPRA similar to theactivities found for the NPRA ligands ANP and BNP. Although antibodiesspecific for NPRA could be selected from the library, none of theantibodies induced activation of NPRA on their own. It may be possiblethat the binding site of the cyclic peptides ANP and BNP on NPRA isstructurally not accessible by a Fab with a mass more than 10 timesbigger than the peptides.

As an alternative, the next sets of selections were performed againstthe receptor-ligand complex. These selections succeeded inidentification of a binder, which specifically recognizes this complex.This antibody, designated 5064, stabilizes the receptor-ligand complexand enhances the potency of ANP or BNP in NPRA dependent cGMPproduction. This antibody now may be used in a variety of therapeuticapplications in which the effects of ANP and/or BNP or any other NPRAagonist are desired. In particular, the potentiating antibodiesidentified herein will be particularly useful in boosting the activitiesof natriuretic peptides. According to data presented herein the 5064antibody is able to decrease the dissociation of ANP from cellular NPRA.

The antibody 5064 was then subjected to affinity maturation in H-CDR2and L-CDR3, which resulted not only in significantly increased bindingto the receptor-ligand complex on cells, but also to increasedbiological activity. Thus in the presence of sub-optimal concentrationsof natriuretic peptides the matured antibodies increased the cGMPresponse stronger than the parental antibody. In conclusion, we can seea clear correlation of antibody affinity and biological activity. Newcombinations of matured L- and H-chains even resulted in a furtherimproved activity of the antibodies. Of interest is the fact that thefive most potent matured antibodies were all optimized in the L-CDR3,while H-CDR2 matured binders showed weaker activities. Maybe the L-CDR3provides crucial binding sites to the receptor, so that optimization ofL-CDR3 has a greater effect than optimization of H-CDR2. In the H-CDR2sequences only a few residues of the parental sequence were changed,which indicates that the H-CDR2 may also be important in binding butdoes not allow major modification of its sequence.

The data presented herein show that affinity maturation starting withonly one candidate can be successful. For the first time a potentiatingantibody was isolated from the HuCAL GOLD® library, which, however, wasnot agonistic by itself, but strongly amplifies the activity of thenatural peptide ligands.

Methods of Making the Antibodies

The sequence of the identified nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes beadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often bases coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Once isolated, the DNA encoding the various portions of the antibody maybe operably linked to expression control sequences or placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,human embryonic kidney cells (HEK), or myeloma cells that do nototherwise produce immunoglobulin protein, to direct the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies is well known in the art.

Expression control sequences refer to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included.

The invention also provides isolated nucleic acids encoding specificbinding agents or antibodies of the invention, optionally operablylinked to control sequences recognized by a host cell, vectors and hostcells comprising the nucleic acids, and recombinant techniques for theproduction of the specific binding agents or antibodies, which maycomprise culturing the host cell so that the nucleic acid is expressedand, optionally, recovering the specific binding agent or antibody fromthe host cell culture or culture medium.

Many vectors are known in the art. Vector components may include one ormore of the following: a signal sequence (that may, for example, directsecretion of the specific binding agent or antibody), an origin ofreplication, one or more selective marker genes (that may, for example,confer antibiotic or other drug resistance, complement auxotrophicdeficiencies, or supply critical nutrients not available in the media),an enhancer element, a promoter, and a transcription terminationsequence, all of which are well known in the art.

Suitable host cells include prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis, Pseudomonas, andStreptomyces. In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forspecific binding agent-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such as Pichia, e.g.P. pastoris, Schizosaccharomyces pombe: Kluyveromyces, Yarrowia;Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces such asSchwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium. Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated specific bindingagent or antibody are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells. Numerous baculoviral strainsand variants and corresponding permissive insect host cells from hostssuch as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), andBombyx mori have been identified. A variety of viral strains fortransfection of such cells are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

However, typical recombinant protein production employs mammalian cells,and propagation of mammalian cells in culture (tissue culture) hasbecome routine procedure. Examples of useful mammalian host cell linesare Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61),DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub etal., Proc. Natl. Acad. Sci. USA 77: 4216, 1980); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, [Graham etal., J. Gen Virol. 36: 59, 1977]; baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243, 1980);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanhepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562. ATCCCCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383: 44, 1982);MRC 5 cells or FS4 cells.

Host cells are transformed or transfected with the nucleic acids orvectors that encode NPRA specific antibodies of the invention arecultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. In addition, novel vectors andtransfected cell lines with multiple copies of transcription unitsseparated by a selective marker are particularly useful and preferredfor the expression of specific binding agents or antibodies.

The host cells transformed with nucleic acids that encode for thedesired antibodies of the invention can be cultured in a variety ofcommonly available culture media, such as for example, media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics,trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

When the host cells are cultured, the antibody or portions thereof canbe produced intracellularly, in the periplasmic space, or directlysecreted into the medium. If the specific binding agent or antibody isproduced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, is removed, for example, bycentrifugation or ultrafiltration.

The antibody composition can then be purified using, for example,hydroxylapatite chromatography, cation or anion exchange chromatography,or preferably affinity chromatography, using the antigen of interest orprotein A or protein G as an affinity ligand. The matrix to which theaffinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled poreglass or poly(styrenedivinyl)benzene allow for faster flow rates andshorter processing times than can be achieved with agarose. Where thespecific binding agent or antibody comprises a CH3 domain, the BakerbondABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein such as ethanol precipitation, ReversePhase HPLC, chromatofocusing. SDS-PAGE, and ammonium sulfateprecipitation are also possible depending on the specific binding agentor antibody to be recovered.

Antibody Derivatives and Variants

The antibodies of the invention could be derivatized. For example, it ispossible to insert amino and/or carboxy terminal fusions of varyingamino acid lengths as well as intra-sequence insertions of single ormultiple amino acid residues. Examples of terminal insertions include aspecific binding agent or antibody with an N-terminal methionyl residueor the specific binding agent or antibody (including antibody fragment)fused to an epitope tag or a salvage receptor epitope. Other insertionalvariants of the specific binding agent or antibody molecule include thefusion to a polypeptide which increases the serum half-life of thespecific binding agent or antibody, e.g. at the N-terminus orC-terminus. Other exemplary mutations that can be prepared includemutations in IgG4 which prevent chain exchange. The mutations can beformed as described in e.g., Marijn et al. Science 317:1554, 2007. Thatreference describes describe a posttranslational modification that leadsto anti-inflammatory activity of antibodies of immunoglobulin G, isotype4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab armsby swapping a heavy chain and attached light chain (half-molecule) witha heavy-light chain pair from another molecule, which results inbispecific antibodies. Mutagenesis studies have shown that the thirdconstant domain is critical for this activity. The impact of IgG4 Fabarm exchange was confirmed in vivo in a rhesus monkey model withexperimental autoimmune myasthenia gravis. IgG4 Fab arm exchange issuggested to be an important biological mechanism that provides thebasis for the anti-inflammatory activity attributed to IgG4 antibodies.Mutating the third constant domain of IgG4 in order to prevent chainexchange in the antibodies described herein is particularly useful.

Examples of epitope tags include the flu HA tag polypeptide and itsantibody 12CA5 [Field et al., Mol. Cell. Biol. 8: 2159, 1988]; the c-myctag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan etal., Mol. Cell. Biol. 5: 3610, 1985]: and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody [Paborsky et al., ProteinEngineering, 3:547, 1990]. Other exemplary tags are a poly-histidinesequence, generally around six histidine residues, that permitsisolation of a compound so labeled using nickel chelation. Other labelsand tags, such as the FLAG®tag (Eastman Kodak, Rochester, N.Y.) are wellknown and routinely used in the art.

The term “salvage receptor binding epitope” refers to an epitope of theFc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the specific bindingagent or antibody molecule removed and a different residue inserted inits place. Substitutional mutagenesis within any of the hypervariable orCDR regions or framework regions is contemplated. Conservativesubstitutions are particularly contemplated. Conservative substitutionsinvolve replacing an amino acid with another member of its class.Non-conservative substitutions involve replacing a member of one ofthese classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the specificbinding agent or antibody to improve its stability (particularly wherethe antibody is an antibody fragment such as an Fv fragment).

Variants of the antibodies described herein can be produced that willhave a modified glycosylation pattern relative to the parent antibody,for example, deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the specific binding agent or antibody.

Glycosylation of polypeptides including antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. The presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. Thus, N-linkedglycosylation sites may be added to a specific binding agent or antibodyby altering the amino acid sequence such that it contains one or more ofthese tripeptide sequences. O-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. O-linkedglycosylation sites may be added to a specific binding agent or antibodyby inserting or substituting one or more serine or threonine residues tothe sequence of the original specific binding agent or antibody.

Cysteine residue(s) may be removed or introduced in the Fc region,thereby eliminating or increasing interchain disulfide bond formation inthis region. The homodimeric specific binding agent or antibody thusgenerated may have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191, 1992 andShopes, B. J. Immunol. 148: 2918, 1992. Homodimeric specific bindingagents or antibodies may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al., Cancer Research 53:2560,1993. Alternatively, a specific binding agent or antibody can beengineered which has dual Fe regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3: 219, 1989.

Sequences within the CDR can cause an antibody to bind to MHC Class IIand trigger an unwanted helper T-cell response. A conservativesubstitution can allow the specific binding agent or antibody to retainbinding activity yet reduce its ability to trigger an unwanted T-cellresponse.

Modifications of the antibodies to increase serum half-life also maydesirable, for example, by incorporation of or addition of a salvagereceptor binding epitope (e.g., by mutation of the appropriate region orby incorporating the epitope into a peptide tag that is then fused tothe specific binding agent or antibody at either end or in the middle,e.g., by DNA or peptide synthesis) (see, e.g., WO96/32478) or addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers.

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the specificbinding agent or antibody or fragment. Even more preferably, three ormore residues from one or two loops of the Fc domain are transferred.Still more preferred, the epitope is taken from the CH2 domain of the Feregion (e.g., of an IgG) and transferred to the CH1, CH3, or VH region,or more than one such region, of the specific binding agent or antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the CL region or VL region, or both, of the specificbinding agent or antibody fragment. These techniques for modifyingantibodies using Fc variants and their interaction with the salvagereceptor are well known to those of skill in the art and have beendescribed e.g., in WO 97/34631 and WO 96/32478.

Other sites of the constant region have been identified that areresponsible for complement dependent cytotoxicity (CDC), such as the C1qbinding site and/or the antibody-dependent cellular cytotoxicity (ADCC)[see, e.g., Mol. Immunol. 29:633, 1992; Shields et al., J. Biol. Chem.,276:6591, 2001, incorporated by reference herein in its entirety].Mutation of residues within Fc receptor binding sites can result inaltered (i.e. increased or decreased) effector function, such as alteredADCC or CDC activity, or altered half-life. As described above,potential mutations include insertion, deletion or substitution of oneor more residues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentsubclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Covalent modifications of the antibody are also contemplated. Suchcovalent modifications may be made by chemical synthesis or by enzymaticor chemical cleavage of the antibody, if applicable. Other types ofcovalent modifications can be introduced into the specific binding agentor antibody by reacting targeted amino acid residues with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the specificbinding agent or antibody in a host cell that has glycosylationcapabilities for N- or O-linked glycosylation. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine,(b) free carboxyl groups, (c) free sulfhydryl groups such as those ofcysteine, (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Thesemethods are described in WO87/05330 published 11 Sep. 1987, and in Aplinand Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the specific bindingagent or antibody may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the specific binding agentor antibody to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the specific binding agent orantibody intact. Chemical deglycosylation is described by Hakimuddin, etal., Arch. Biochem. Biophys. 259:52 1987 and by Edge et al. Anal.Biochem., 118:131, 1981. Enzymatic cleavage of carbohydrate moieties ona specific binding agent or antibody can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol. 138: 350, 1987.

Another type of covalent modification of the antibody comprises linkingit to one of a variety of nonproteinaceous polymers, e.g., polyethyleneglycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylatedsorbitol, polyoxyethylated glucose, polyoxyethylated glycerol,polyoxyalkylenes, or polysaccharide polymers such as dextran. Suchmethods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Methods of Therapy

“Treatment” is an intervention performed with the intention ofpreventing the development, progression, or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude subjects that already have symptoms of the disorder as well asthose who have been diagnosed as likely to develop the disorder andhence in whom the disorder is to be prevented. The phrase “treatment”may include ameliorating, suppressing, eradicating, reducing theseverity of, decreasing the frequency of incidence of, preventing,reducing the risk of, and/or delaying the onset of the condition.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic NPRA antibody thatprolongs the biological effects of ANP and/or BNP through the action ofthe NPRA receptor. In particular aspects, these methods are effectivetreatments for a variety of cardiovascular conditions. Sucheffectiveness may be realized in, for example, efficacy, potency, dosingrequirements, and/or reduced side effects. The term “cardiovascularcondition” is used broadly in this application, and includes, forexample, hypertension (including resistant hypertension and pulmonaryhypertension), heart failure (such as chronic heart failure (i.e.,“CHF”), or heart failure following myocardial infarction), arrhythmia,diastolic dysfunction (such as left ventricular diastolic dysfunction,diastolic heart failure, or impaired diastolic filling), systolicdysfunction, ischemia (such as myocardial ischemia), cardiomyopathy(such as hypertrophic cardiomyopathy and dilated cardiomyopathy), suddencardiac death, myocardial fibrosis, vascular fibrosis, impaired arterialcompliance, myocardial necrotic lesions, vascular damage in the heart,vascular inflammation in the heart, myocardial infarction (“MI”)(including both acute post-MI and chronic post-MI conditions), coronaryangioplasty, left ventricular hypertrophy, decreased ejection fraction,coronary thrombosis, cardiac lesions, vascular wall hypertrophy in theheart, endothelial thickening, myocarditis, coronary artery disease(such as fibrinoid necrosis of coronary arteries), and atherosclerosis.

Administering the antibodies provides an effective treatment for avariety of conditions that are associated (either directly orindirectly) with hypertension, heart failure, and/or othercardiovascular conditions. Such secondary conditions include, forexample, renal dysfunctions, cerebrovascular diseases, vascular diseasesgenerally, retinopathy, neuropathy (such as peripheral neuropathy),edema, endothelial dysfunction, and insulinopathy (includingcomplications arising from insulinopathy). Examples of renaldysfunctions include glomerulosclerosis, end-stage renal disease, acuterenal failure, diabetic nephropathy, reduced renal blood flow, increasedglomerular filtration fraction, proteinuria, decreased glomerularfiltration rate, decreased creatine clearance, microalbuminuria, renalarteriopathy, ischemic lesions, vascular damage in the kidney, vascularinflammation in the kidney, and malignant nephrosclerosis (such asischemic retraction, thrombonecrosis of capillary tufts, arteriolarfibrinoid necrosis, and thrombotic microangiopathic lesions affectingglomeruli and microvessels). Examples of cerebrovascular diseasesinclude stroke. Examples of vascular diseases include thromboticvascular disease (such as mural fibrinoid necrosis, extravasation andfragmentation of red blood cells, and luminal and/or mural thrombosis),proliferative arteriopathy (such as swollen myointimal cells surroundedby mucinous extracellular matrix and nodular thickening),atherosclerosis, decreased vascular compliance (such as pathologicalvascular stiffness and/or reduced ventricular compliance), andendothelial dysfunction. Examples of edema include peripheral tissueedema and lung congestion. Examples of insulinopathies include insulinresistance, Type I diabetes mellitus, Type II diabetes mellitus, glucosesensitivity, pre- and diabetic syndrome X.

Thus, in some embodiments, the pathological condition comprises acardiovascular disease, renal dysfunction, edema, a cerebrovasculardisease, or an insulinopathy. In other embodiments, the condition to betreated is a cardiovascular disease, stroke, or type II diabetes. Instill other embodiments, the condition to be treated is hypertension,heart failure, left ventricular hypertrophy, or stroke. In still otherembodiments, the condition to be treated is a cardiovascular disease. Insome other aspects, the condition to be treated is hypertension. Instill other embodiments, the condition to be treated is heart failure,arrhythmia, diastolic dysfunction, systolic dysfunction, ischemia,cardiomyopathy, sudden cardiac death, myocardial fibrosis, vascularfibrosis, impaired arterial compliance, myocardial necrotic lesions,vascular damage in the heart, myocardial infarction, left ventricularhypertrophy, decreased ejection fraction, vascular wall hypertrophy inthe heart, or endothelial thickening. The heart failure may be acuteheart failure, acute post-myocardial-infarction heart failure, chronicheart failure, chronic post-myocardial-infarction heart failure,hypertension-driven heart failure, sudden cardiac death, vascularinflammation in the heart. The condition to be treated may be coronaryangioplasty, coronary thrombosis, cardiac lesions, myocarditis, coronaryartery disease, such as fibrinoid necrosis of coronary arteries. Inother aspects, the condition to be treated is renal dysfunction. Instill other embodiments, the condition to be treated is acerebrovascular disease.

In exemplary combination protocols the subject is dosed with a firstcomposition comprising the antibody composition and a second compositioncomprising the addition therapeutic agent for the treatment of thedisorder. The first and second compositions together form atherapeutically-effective treatment for the targeted condition(s). Itshould be recognized that the specific dose level and frequency ofdosing for the antibody and other therapeutic agents will depend on avariety of factors including, for example, the particular combination ofagents selected: the activity, efficacy, pharmacokinetic, and toxicologyprofiles of the particular therapeutic agents used (including suchprofiles when the agents are used in combination); the age, weight,general health, sex, and diet of the patient; the frequency ofadministration; the rate of excretion; the condition(s) being treated;the severity of the condition(s) being treated; whether a drug deliverysystem is used; the form, route, and frequency of administration; andwhether other pharmaceutically-active compounds also are beingadministered. Thus, the dosage regimen actually employed may varywidely, and therefore may deviate from the preferred dosage regimens setforth in this patent.

The total daily dose of each drug generally may be administered to thepatient in a single dose, or in proportionate multiple subdoses.Subdoses typically are administered from 2 to about 6 times per day, andmore typically from 2 to about 4 times per day. Doses may be in animmediate-release form or sustained-release form effective to obtaindesired results. It should be recognized that, although the dosingfrequency for the therapeutic agents in this invention is typicallydaily or multiple times per day, this invention also contemplates dosingregimens wherein the preferred period between administration of one ormore of the therapeutic agents is greater than 24 hours. In suchembodiments, the dosing frequency may be, for example, every 36 hours,every 48 hours, every 72 hours, weekly, or monthly.

In the combination therapies contemplated, the administration maycomprise administering the antibody and the second agent in asubstantially simultaneous manner using either a single formulation(e.g., a single capsule) having a fixed ratio of the therapeutic agents,or separate formulations (e.g., multiple capsules) that each comprise atleast one of the therapeutic agents. Such administration also maycomprise administering the antibody and other therapeutic agent atdifferent times in separate formulations. This may include, for example,administering the components of the combination in a sequential manner.Or it may include administering one component multiple times between theadministrations of another component. Or it may include administeringtwo components at the same time, while also separately administeringanother portion at least one of those components at a different time aswell. Or it may include administering the two components sequentiallyfor a two-step effect. Where the components of the combination are dosedseparately, the time period between the dosing of each component mayrange from a few minutes to several hours or days, and will depend on,for example, the properties of each component (e.g., potency,solubility, bioavailability, half-life, and kinetic profile), as well asthe condition of the patient.

Dosage and dosage-frequency optimization (to the extent desirable) maybe determined in trials. It should be recognized that multiple doses perday typically may be used to increase the total daily dose, if desired.

Dosing of the first and second compositions can be determined andadjusted based on measurement of parameters that would be known to oneskilled in the art. Non-limiting examples of such parameters generallyinclude blood pressure, pulmonary capillary wedge pressure orappropriate surrogate markers (such as cGMP, natriuretic peptides,endothelins, and other surrogate markers). Blood pressure, pulmonarycapillary wedge pressure and/or surrogate marker levels afteradministration of the combination therapy can be compared against thecorresponding baseline levels before administering the therapy todetermine efficacy of the present method and titrated as needed.Non-limiting examples of surrogate markers useful in the method aresurrogate markers for renal and cardiovascular disease.

It should be recognized that it is often preferred to start dosing thetherapeutic agents of the combination at an intermediate levels(particularly an intermediate levels falling within the above-describedpreferred dosage ranges), and then titrate up or down, depending onobserved efficacy and side-effects. In many embodiments, treatment iscontinued as necessary over a period of several weeks to several monthsor years until the condition(s) has been controlled or eliminated.Patients undergoing treatment with the antibodies disclosed herein canbe routinely monitored by a wide variety of methods known in the art fordetermining the effectiveness of a treatment for the particularcondition being treated. This may include, for example, blood pressure,echocardiography; MRI; monitoring C-reactive protein, brain natriureticpeptides (“BNP”), fibrinogen levels, and pro-inflammatory molecule(e.g., TNFα, MMP-2, MMP-3, MMP-13, etc.) and cGMP levels in thebloodstream; and, for kidney-related diseases, it also may include, forexample, monitoring the urea appearance rate (“UAR”). Kidney functioncal also be measured using creatinine clearance and cystatin levelsusing methods known to those of skill in the art. Continuous analysis ofsuch data permits modification of the treatment regimen during therapyso that optimal effective amounts of each type of therapeutic agent areadministered at any time, and so that the duration of treatment can bedetermined as well. In this way, the treatment regimen/dosing schedulecan be rationally modified over the course of therapy so that the lowestamount of each therapeutic agent that together exhibit satisfactoryeffectiveness is administered, and so that administration is continuedonly so long as is necessary to successfully treat the condition.

The antibody treatment and/or the combination therapies of thisinvention may be administered prophylactically, before a diagnosis of acardiovascular condition (or associated condition), and to continueadministration of the combination during the period of time the subjectis susceptible to the condition. Individuals with no remarkable clinicalpresentation, but that are nonetheless susceptible to pathologiceffects, therefore can be placed on a prophylactic dose of thecombination. Such prophylactic doses may, but need not, be lower thanthe doses used to treat the specific pathogenic effect of interest.

In some embodiments of this invention, cardiac pathologies areidentified, and an effective dosing and frequency determined, based onblood concentrations of natriuretic peptides. Elevated natriureticpeptide levels in the blood, particularly blood BNP levels, generallyare observed in subjects under conditions of blood volume expansion andafter vascular injury such as acute myocardial infarction and remainelevated for an extended period of time after the infarction. (Uusimaaet al., Int. J. Cardiol, 69:5, 1999. A decrease in natriuretic peptidelevel relative to the baseline level measured before administration of atherapy (antibody alone or antibody in combination with another therapy)of this invention indicates a decrease in the pathologic effect mediatedby the therapy, and, therefore, provides a correlation with inhibitionof the pathologic effect. Blood levels of the desired natriureticpeptide level therefore can be compared against the correspondingbaseline level before administration of the therapy to determineefficacy of the present method in treating the pathologic effect. Basedon such natriuretic peptide level measurements, dosing of thecombination can be adjusted to reduce the cardiovascular pathologiceffect. Efficacy of the therapeutic agent and determination of theappropriate dosing can also be based on circulating and urinary cGMPLevels. An increased plasma level of cGMP parallels a fall in pulmonarycapillary wedge pressure. Increased urinary excretion of cGMP can becorrelated with the natriuresis.

In some embodiments, a therapy of this invention is administered at adosage and frequency effective to cause a statistically-significantdecrease in tissue or circulating C-reactive protein (CRP) levels.

In some embodiments, a therapy of this invention is administered to apatient having an ejection fraction of less than about 45%, particularlyless than about 40%, and even more particularly less than about 30%. Insuch embodiments, the therapy preferably is administered at a dosage andfrequency effective to cause a statistically-significant increase (orpreserve, or at least partially preserve) left ventricular ejectionfraction. In other embodiments, the therapy is administered in an amounteffective to achieve hemodynamic improvements such as improved cardiacoutput, pulmonary capillary wedge pressure. The therapy also may beuseful in producing a decrease in infarct size post-MI.

In some embodiments, a therapy of this invention is administered at adosage and frequency effective to cause a statistically-significantincrease (or preserve, or at least partially preserve) stroke volume.

In some embodiments, a therapy of this invention is administered at adosage and frequency effective to cause a statistically-significantdecrease in left ventricular end systolic area, end diastolic area, endsystolic volume, or end diastolic volume.

In some embodiments, a therapy of this invention is administered at adosage and frequency effective to cause a statistically-significantdecrease in left ventricular mass.

In some embodiments, a therapy of this invention is administered at adosage and frequency effective to cause a statistically-significantdecrease in interstitial collagen fraction in the heart (which can bemonitored by, for example, measuring collagen markers or measuring thestiffness of the heart using, for example, an echocardiogram).

In some embodiments, a therapy of this invention is administered basedon the presence of myocardial infarction or heart failure or leftventricular hypertrophy. Left ventricular hypertrophy can be identifiedby echo-cardiogram or magnetic resonance imaging and used to monitor theprogress of the treatment and appropriateness of the dosing.

For the treatment of hypertension, the subject is typically firstidentified as normotensive, borderline hypertensive, or hypertensivebased on blood pressure determinations. For humans, in particular, sucha determination may be achieved using a seated cuff mercurysphygmomanometer. Individuals may be deemed normotensive when systolicblood pressure and diastolic blood pressure are less than about 125 mmHg and less than about 80 mm Hg, respectively; borderline hypertensivewhen systolic blood pressure and diastolic blood pressure are in therange of from about 125 to about 140 mm Hg and from about 80 to about 90mm Hg, respectively; and hypertensive when systolic blood pressure anddiastolic blood pressure are greater than about 140 mm Hg and 90 mm Hg,respectively. As the severity of the hypertensive condition increases,the preferred dose of at least one component of the therapy typicallyincreases. Based on post-administration blood pressure measurement, thedoses of the components of the combination may be titrated. After aninitial evaluation of the subject's response to the treatment, the dosesmay be increased or decreased accordingly to achieve the desired bloodpressure lowering effect.

Dosing and frequency to treat pathologies of renal function can bedetermined and adjusted based on, for example, measurement ofproteinuria, microalbuminuria, decreased glomerular filtration rate(GFR), or decreased creatinine clearance. Proteinuria is identified bythe presence of greater than about 0.3 g of urinary protein in a 24 hoururine collection. Microalbuminuria is identified by an increase inassayable urinary albumin. Based upon such measurements, dosing of thedosing and frequency of a combination of this invention can be adjustedto ameliorate a renal pathologic effect.

Neuropathy, especially peripheral neuropathy, can be identified by, anddosing and frequency adjustments based on, neurologic exam of sensorydeficit or sensory motor ability. Retinopathy can be identified by, anddosing and frequency adjustments based on, ophthalmologic exam.

Animal Models for Monitoring Therapy

Various animal models are available for testing the therapeuticcompositions of the invention. For example, the antibody preparationseither alone or in combination with other recognized treatments forheart failure may be tested in rat models for spontaneously hypertensiveheart failure. Such a rat model has been described in the art. Heyen etal., “Structural, functional, and molecular characterization of the SHHFrat model of heart failure”, Am. J. Physiol., 283:H1775, 2002(incorporated by reference into this patent). This model may be used asdescribed below to evaluate the therapeutic potential of the antibodiesand or combination therapies contemplated herein.

For example, lean, male SHHF rats (Genetic Models Inc., Indianapolis,Ind.) are used as the test models and age-matched Sprague-Dawley (SD)rats (Charles River Labs, Raleigh, N.C.) are used as controls. Allanimals are acclimated to their environment, e.g., housed in a roomlighted for 12 hours per day at an ambient room temperature.

Another model that can be used is the volume expanded hypertensive ratmodel (also known as the aldosterone/salt rat model) which has beendescribed in the art. See, e.g., Rocha, R., et al., Am. J. Physiol.Heart Circ. Physiol., 283: H1802, 2002. See also, Blasi, E. R., et al.,Kidney International, 63: 1791, 2003. See also, PCT Patent PublicationNo. WO 01/95893.

Following acclimation, unnephrectomized rats are given 1% NaCl drinkingwater and infused subcutaneously with aldosterone (0.5 g/kg/hr) via anAlza osmotic pump, Model 2002. These rats are assigned to one of thefollowing treatment groups: (1) rats receiving no treatment; (2) ratsreceiving a second therapeutic agent of interest at a dosing ofinterest, (3) rats receiving an antibody of interest at a dosing ofinterest, and (4) rats receiving a co-administration of the aldosteroneantagonist at a dosing of interest and the antibody at a dosing ofinterest. The treatments continued for 3 weeks. Over that period, bloodpressure and heart rate are evaluated continuously by telemetry via animplanted transmitter connected to a pressure transducer cannulated tothe abdominal aorta. The blood pressure and heart rate data is averagedover 24-hour periods.

The stroke prone spontaneously hypertensive rat (SHR-SP) model has beendescribed in the art. See, e.g., Rocha, R., et al., Trends in Endocrin.& Met., 12: 308, 2001.

The study is conducted over a defined period of time, e.g., 12 weeks,with measurements and samples taken at baseline, and at set intervalsthereafter (e.g., after 4, 8, and 12 weeks). Following acclimation,baseline measurements are performed, and 1 week later, the rats areassigned to one of the following treatment groups after being randomizedbased on genotype: (1) rats receiving no treatment; (2) rats receivingthe antibody at a dose of interest, (3) rats receiving a second agent ofinterest at a dose of interest, and (4) rats receiving aco-administration of the antibody at a dose of interest and the secondagent at a desired dose of interest.

The rats are monitored for transthoracic echocardiography. See Heyen, J.R. R., et al. The examinations are performed at baseline, and after 4,9, and 13 weeks of treatment during the progression of heart failure.During these examinations, each animal is lightly anesthetized, thechest is shaved, and echocardiograms are obtained.

Intra-ventricular systolic blood pressure is measured following 12 weeksof treatment. During this analysis, each animal is anesthetized and theright common carotid artery is cannulated with a Millar cathetertransducer (Millar, Houston, Tex.) passed under constant pressure intothe left ventricle. Data is collected every 10 seconds for 3 minutes andanalyzed using a HPA-210 heart performance analyzer (Micro-Med,Louisville, Ky.).

Alternatively, tail-cuff systolic blood pressure is analyzednon-invasively at baseline, and after 6 and 12 weeks of treatment usingthe Visitech BP-2000 Blood Pressure Analysis System (Visitech Systems,Apex, N.C.). Six measures are taken for each animal and averaged for amean SBP reading.

Serum electrolytes are analyzed using a Hitachi 912 automated diagnosticclinical chemistry analyzer (Roche Diagnostics Corp., Indianapolis,Ind.) according to standard procedures.

At the end of the experiment, each animal is anesthetized and weighed.The abdominal cavity is opened to expose the abdominal aorta. An18-gauge needle is then inserted into the abdominal aorta, and theanimals are exsanguinated. The resulting blood is immediatelytransferred into serum collection tubes and the samples are thencentrifuged for 15 min at 3,000 rev/min at 4° C. to form a serum thatis, in turn, collected and frozen at −80° C. until further analysis.

Following exsanguination, the heart is isolated, removed, rinsed in coldPBS (Gibco, Gaithersburg, Md.), blotted dry, and weighed. Tibia also areremoved (documented by X-ray analysis), and the length is determinedusing calipers. The observed heart weight is then normalized to tibiallength (HW/TL). A 6-mm section is cut transversely through the middle ofthe heart and placed into 10% neutral-buffered formalin for 24 hr,followed by 70% alcohol until embedded into paraffin. The remainingapical portion of the heart is snap frozen in liquid nitrogen and storedat −80° C. for molecular analysis.

Urinary proteinuria is determined by using the Bio-Rad protein dyereagent (Hercules, Calif.). The assay is modified to a 96-well plateformat according to the manufacturer's instructions.

During this experiment, the groups of rats are compared with respect to,for example, systolic blood pressure, ejection fraction, stroke volume,left ventricular end diastolic area, left ventricular end systolic area,left ventricular end diastolic volume, left ventricular end systolicvolume, urinary protein, TNFα in the serum and heart tissue, leftventricular mass (absolute and normalized to tibial length), plasmaosteopontin, and MMP levels and activity.

Another rat model that has been commonly described in the art and couldbe used for testing antibody preparations alone or in combination withstandard treatments is a coronary artery ligation model (eg Raya, et.al., Circ Res 64:330, 1989). Adult male Sprague-Dawley rats undergoexperimental myocardial infarction (MI) by standard techniques in whichthe animals are anesthetized and a left thoracotomy is performed, theheart is expressed from the thorax, and a ligature is placed around theproximal left coronary artery. The heart is then returned to the chestand the thorax closed. Following recovery, the rats are treated withantibody alone or in combination with a second agent of interest asoutlined above. After 3-5 weeks of treatment the animals areanesthetized and cardiac function is measured as outlined above.

Canine models of chronic heart failure have also been described in theart. See, e.g., Suzuki, G., “Effects of Long-Term Monotherapy WithEplerenone, a Novel Aldosterone Blocker, on Progression of LeftVentricular Dysfunction and Remodeling in Dogs with heart failure”,Circulation, vol. 106, pp. 2967-2972 (Dec. 3, 2002) (incorporated byreference into this patent). See also, Sabbah, H. N., et al., “A caninemodel of chronic heart failure produced by multiple sequential coronarymicroembolizations”, Am. J. Physiol., 260: H1379 1991 (incorporated byreference into this patent). This model can be used to evaluate thetherapies contemplated herein.

In this study, mongrel dogs undergo serial coronary microembolizationsto produce heart failure. Embolizations are performed 1 to 3 weeksapart, and are discontinued when left ventricular ejection fraction is30% to 40%. Microembolizations are performed during cardiaccatheterization under general anesthesia and sterile conditions.Anesthesia consists of a combination of intravenous injections ofoxymorphone (0.22 mg/kg), diazepam (0.17 mg/kg), and sodiumpentobarbital (150 to 250 mg to effect).

Two weeks after the last microembolization, the dogs undergo apre-randomization left and right heart catheterization. One day later,the dogs are randomized, and then assigned to one of the followingtreatment groups: (1) dogs receiving no treatment; (2) dogs receiving anantibody of interest at a dosing of interest, (3) dogs receiving acombination therapy of the invention, and (4) dogs receiving the secondtherapeutic agent used in the combination therapy at a dosing ofinterest. This treatment is continued for 3 months. Final hemodynamicand angiographic measurements are made at the end of the 3 months. Whileunder anesthesia, the each dog's chest is opened, the heart is removed,and tissue is prepared for biochemical and histological evaluations.

During this experiment, the groups of dogs are compared with respect to,for example, changes in left ventricular ejection fraction;end-diastolic volume; end-systolic volume; peak left ventricular +dP/dt;peak left ventricular −dP/dt; pulmonary artery pressure; the timeconstant of isovolumic relaxation, r, left ventricular end-diastolic andend-systolic axes ratios (which, in turn, indicate changes in leftventricular chamber sphericity); left ventricular end-diastolic wallstress; body weight; heart weight (normalized with body weight); leftventricular wall thickness; Na+, K+, BUN, and creatinine; mean aorticpressure; and heart rate. Comparisons also are made with respect to, forexample, cardiac myocyte cross-sectional area (which, in turn, is ameasure of cell hypertrophy), volume fraction of interstitial fibrosis,and volume fraction of replacement fibrosis, and capillary density,gelatinase activity, and transcription of basic fibroblast growthfactor.

Another exemplary model that may be used to monitor treatment is acanine model of pacing induced heart failure. This model is well knownto those of skill and is described in for example in Katsuya, et. al., JCardiovasc Pharmacol 43: 860 2004. To induce heart failure by rapidright ventricular pacing, a modified multiprogrammable pacemaker(Medtronics, Inc.) is implanted in healthy adult male, mongrel dogs.After full recovery from the instrumentation (10 to 14 days aftersurgery), the animals are subjected to rapid ventricular pacing at 240bpm. On the eighth day of pacing the dogs are assigned to the one of thefollowing treatment protocols: (1) dogs receiving no treatment; (2) dogsreceiving an antibody of interest at a dosing of interest, (3) dogsreceiving a combination therapy of the invention, and (4) dogs receivingthe second therapeutic agent used in the combination therapy at a dosingof interest. After 4 weeks of pacing, the pacemaker was turned off andthe animals were allowed to equilibrate for 30 to 40 minutes.Hemodynamic and cardiac measurements are then done as outlined above.

Pharmaceutical Preparations

The NPRA specific antibodies used in the practice of a method of theinvention may be formulated into pharmaceutical compositions comprisinga carrier suitable for the desired delivery method. Suitable carriersinclude any material which, when combined with the antibody, retains thehigh-affinity binding and ligand potentiating properties of the antibodyand is preferably nonreactive with the subject's immune systems.Examples include, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Exemplary antibody concentrations in the formulation may range fromabout 0.1 mg/ml to about 200 mg/ml or from about 0.1 mg/mL to about 50mg/mL, or from about 0.5 mg/mL to about 25 mg/mL, or alternatively fromabout 2 mg/mL to about 10 mg/mL. An aqueous formulation of the antibodymay be prepared in a pH-buffered solution, for example, at pH rangingfrom about 4.5 to about 6.5, or from about 4.8 to about 5.5, oralternatively about 5.0. Examples of buffers that are suitable for a pHwithin this range include acetate (e.g. sodium acetate), succinate (suchas sodium succinate), gluconate, histidine, citrate and other organicacid buffers. The buffer concentration can be from about 1 mM to about200 mM, or from about 10 mM to about 60 mM, depending, for example, onthe buffer and the desired isotonicity of the formulation.

Tonicity agents to stabilize the antibody may be included in thepharmaceutical formulation. Exemplary tonicity agents include polyols,such as mannitol, sucrose or trehalose. Preferably the aqueousformulation is isotonic, although hypertonic or hypotonic solutions maybe suitable. Exemplary concentrations of the polyol in the formulationmay range from about 1% to about 15% w/v.

A surfactant may also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbate 20, or polysorbate 80) or poloxamers (e.g. poloxamer 188).Exemplary concentrations of surfactant may range from about 0.001% toabout 0.5%, or from about 0.005% to about 0.2%, or alternatively fromabout 0.004% to about 0.01% w/v.

The antibodies also may be formulated with various preservatives (e.g.,benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl.) Ifpreservatives are present, they may be included in the formulation atconcentrations ranging from about 0.1% to about 2%, or alternativelyfrom about 0.5% to about 1%. One or more other pharmaceuticallyacceptable carriers, excipients or stabilizers such as those describedin Remington's The Practice and Science of Pharmacy 21 Edition. (2005)may be included in the formulation provided that they do not adverselyaffect the desired characteristics of the formulation. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed and include; additional bufferingagents; co-solvents; antioxidants including ascorbic acid andmethionine; chelating agents such as EDTA; metal complexes (e.g.Zn-protein complexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's The Practice and Science of Pharmacy 21^(st) Edition.(2005)), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides: proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,maltose, or dextrins; chelating agents such as EDTA; sugars such assucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

In one embodiment, a suitable formulation of the claimed inventioncontains an isotonic buffer such as a phosphate, acetate, or TRIS bufferin combination with a tonicity agent such as a polyol, Sorbitol, sucroseor sodium chloride which tonicifies and stabilizes. One example of sucha tonicity agent is 5% Sorbitol or sucrose. In addition, the formulationcould optionally include a surfactant such as to prevent aggregation andfor stabilization at 0.01 to 0.02% wt/vol. The pH of the formulation.may range from 4.5-6.5 or 4.5 to 5.5. Other exemplary descriptions ofpharmaceutical formulations for antibodies may be found in US2003/0113316 and U.S. Pat. No. 6,171,586.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.The compositions of the invention may be sterilized by conventional,well known sterilization techniques. For example, sterilization isreadily accomplished by filtration through sterile filtration membranes.The resulting solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile solution prior to administration. Methods offreeze-drying polypeptides for long term storage are well known.(Williams and Polli, Journal of Parenteral Science and Technology,38:48, 1984. The lyophilization cycle involves freezing, primary drying,and secondary drying. The process produces a material known as alyophilized cake. Thereafter the cake can be reconstituted prior to use.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water (typically equivalent to the volume removedduring lyophilization), although dilute solutions of antibacterialagents are sometimes used in the production of pharmaceuticals forparenteral administration; Chen, Drug Development and IndustrialPharmacy, 18: 1311, 1992.

Excipients have been noted in some cases to act as stabilizers forfreeze-dried products; Carpenter et al., Developments in BiologicalStandardization, 74: 225, 1991. For example, known excipients includepolyols (including mannitol, sorbitol and glycerol); sugars (includingglucose and sucrose); and amino acids (including alanine, glycine andglutamic acid).

In addition, polyols and sugars are also often used to protectpolypeptides from freezing and drying-induced damage and to enhance thestability during storage in the dried state. In general, sugars, inparticular disaccharides, are effective in both the freeze-dryingprocess and during storage. Other classes of molecules, including mono-and di-saccharides and polymers such as PVP, have also been reported asstabilizers of lyophilized products.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions; sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

The specific binding agent or antibody is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions include intravenous,intraarterial, intraperitoneal, intramuscular, intradermal orsubcutaneous administration. In addition, the specific binding agent orantibody is suitably administered by pulse infusion, particularly withdeclining doses of the specific binding agent or antibody. Preferablythe dosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Other administration methods are contemplated,including topical, particularly transdermal, transmucosal, rectal, oralor local administration e.g. through a catheter placed close to thedesired site. Most preferably, the specific binding agent or antibody ofthe invention is administered intravenously in a physiological solutionat a dose ranging between 0.01 mg/kg to 100 mg/kg at a frequency rangingfrom daily to weekly to monthly (e.g. every day, every other day, everythird day, or 2, 3, 4, 5, or 6 times per week), preferably a doseranging from 0.1 to 45 mg/kg, 0.1 to 15 mg/kg or 0.1 to 10 mg/kg at afrequency of 2 or 3 times per week, or up to 45 mg/kg once a month.Another preferred method of administration is through inhalation.

Combination Therapy

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a pathological condition. In thisspecification, the pathological condition generally comprises acardiovascular condition or a condition associated with a cardiovascularcondition. The therapeutic agents of the combination generally may beco-administered in a substantially simultaneous manner, such as, forexample, (a) in a single formulation (e.g., a single capsule) having afixed ratio of active ingredients, or (b) in multiple, separateformulations (e.g., multiple capsules) for each agent. The therapeuticagents of the combination may alternatively (or additionally) beadministered at different times. In either case, the chosen treatmentregimen preferably provides beneficial effects of the drug combinationin treating the condition.

In the context of combination therapy, the phrase“therapeutically-effective” qualifies the amount of each therapeuticagent that will achieve the goal of ameliorating, suppressing,eradicating, reducing the severity of, decreasing the frequency ofincidence of, preventing, reducing the risk of, and/or delaying theonset of a pathological condition.

The treatment of heart disease includes standard treatments such as useof angiotensin II converting enzyme (ACE) inhibitors, β-adrenoceptorinhibitors, and aspirin. Any such standard therapies may be combinedwith therapeutic intervention using the antibodies of the presentinvention. Angiotensin receptor blockers may also be used in patientswho do not tolerate ACEI's, and candesartan has recently been approvedfor use in combination with ACEI's. Aldosterone antagonists such as forexample, eplerenone, also have proven effective in the treatment ofheart failure and hypertension. The antibodies of the invention alsocould be used in combination with statin therapy in heart failure.

The antibodies described herein may be used in combination with renininhibitors, such as for example aliskiren (Tekturna, Rasilez, Novartis).Endothelin antagonists and vasopeptidase inhibitors (dual ACE/neutralendopeptidase (NEP) inhibitors) also may be useful in combinationtherapies. Combination therapies with vasopressin antagonists also maybe useful. Also contemplated for combination therapy are diuretic agentssuch as A1 adenosine receptor antagonists. Other useful agents includebeta blocker agents for the treatment of CHF.

Inotropic agents such as digoxin have long been used to relieve thesymptoms of severe CHF. Milrinone is a PDE3 inhibitor and inotropicagent that is used only for short term treatment of acute CHF becausethis class of agents is proarrhythmiagenic and can have a negativeimpact on survival with chronic use. Cardiac myosin activators also mayprove useful in combination with the therapies of the present invention.Also contemplated for use in the combination therapies of the presentinvention are PDE5 inhibitors e.g., Viagra, Cialis as well as soluble GCactivators/stimulators currently in clinical trials.

Combination of the antibodies of the invention with Nesiritide (hBNP)and other natiuretic peptides (either recombinant or naturally isolated)is particularly contemplated. The antibodies may be combined withNesiritide, Carperitide (ANP, Suntory, now Daiichi-Sankyo), Ularitide(urodilatin, PDL, EKR Therapeutics) and combination of all three areparticularly contemplated.

The phrase “aldosterone antagonist” embraces an agent or compound, or acombination of two or more of such agents or compounds, which counteractthe effect of aldosterone. Such agents and compounds, such asmespirenone, may antagonize the action of aldosterone through apre-receptor mechanism. Other agents and compounds, such asspironolactone and eplerenone, fall generally within a class known asaldosterone receptor antagonists, which bind to mineralocorticoidreceptors to prevent natural ligand activation of post-receptor events.Many suitable aldosterone antagonists are described by, for example,Perez et al. in U.S. Pat. No. 6,410,524 (issued Jun. 25, 2002; filedNov. 5, 1999 as U.S. patent application Ser. No. 09/434,685)(incorporated by reference into this patent).

The aldosterone antagonists used in the methods of the present inventiongenerally are spirolactone-type steroidal compounds as exemplified byspirolactone itself. The epoxy-steroidal aldosterone antagonistcompounds also may be used in the combination therapies contemplatedherein. Of particular interest is the compound eplerenone (also known asepoxymexrenone). Eplerenone is an aldosterone receptor antagonist, andhas a greater specificity for aldosterone receptors than does, forexample, spironolactone. Selection of eplerenone as the aldosteroneantagonist in the present method would generally tend to be beneficialfor reducing certain side-effects, such as, for example, gynecomastia(which tends to occur when less-specific aldosterone antagonists areused).

The term “diuretic” includes, for example, diuretic benzothiadiazinederivatives, diuretic organomercurials, diuretic purines, diureticsteroids (including diuretic steroids having no substantial activity asan aldosterone receptor antagonist), diuretic sulfonamide derivatives,diuretic uracils, etc. Exemplary such compounds include amanozine,amiloride, arbutin, chlorazanil, ethacrynic acid, mannitol,metochalcone, muzolimine, perhexiline, and urea which can be purchasedfrom commercial sources. The diuretic compound also may be abenzothiadiazine derivative, a sulfonamide derivative an organicmercurial diuretic such as mercaptomerin sodium, merethoxylline,procaine, and mersalyl with theophylline. In specific examples thediuretic is amiloride, ethacrynic acid, triamterene,hydrochlorothiazide, chlorothiazide, bumetamide, furosemide, orhydrochlorothiazide.

The present invention further comprises kits that are suitable for usein performing the methods of treatment described above. In oneembodiment, the kit comprises a first dosage form comprising antibody ofthe invention and a second dosage form comprising a second agent for apathological condition (e.g., a cardiovascular condition or a conditionassociated with a cardiovascular condition) in quantities sufficient tocarry out the methods of the present invention. Preferably, the firstdosage form and the second dosage form together comprise atherapeutically-effective amount of the agents for the treatment of thetargeted condition(s).

EXAMPLES

The following examples are offered by way of illustration and are notmeant to be limiting in any way.

Example 1 Generation of Stable Cell Lines Expressing Natriuretic PeptideReceptors

Full-length human NPRA, human NPRB, human NPRC, rhesus monkey NPRA andcanine NPRA sequence containing plasmids are purchased from OriGeneTechnologies, Inc. (Rockville, Md.) or the sequences are synthesized byDNA2.0 then sub-cloned into the pcDNA3.1 mammalian expression vector(Invitrogen Corporation, Carlsbad, Calif.). Insert orientation andnucleotide sequence of each construct is verified by an outside vendor.The pcDNA3.1 NPR clones are transfected using Lipofectamine (Invitrogen)into HEK293 cells where stable cell lines are selected using G418. NPRAand NPRB clones are screened using the ANP- or CNP- (Sigma-Aldrich, St.Louis, Mo.) induced cGMP assay described below. NPRC clones are screenedusing the ¹²⁵I-ANP binding assay outlined below. High cGMP producing orhigh ¹²⁵I-ANP binding clones are expanded in DMEM containing, 100 μg/mLpenicillin/streptomycin, L-glutamine, 400 μg/mL of G418, and 10% fetalbovine serum (Hyclone, Logan, Utah). HEK293T-GCA, rat NPRA expressingcells obtained from Dr. Lincoln Potter (University of Minnesota), aregrown in DMEM containing 100 μg/mL penicillin/streptomycin, L-glutamine,Hygromycin B and 10% fetal bovine serum.

Example 2 Generation of NPRA-Fc Fusion Protein

A. Construction of the NPRA-Fc Fusion Protein

The extracellular domain (ECD) of NPRA is fused to human Fc (gamma 1) toobtain a soluble form of NPRA. To accomplish this, plasmidpcDNA3.1+kappa-leader-MORxx-Fc is first restricted with KpnI (MORxxencoding for unnamed control protein). An oligolinker containing a ClaIsite is inserted into the KpnI site via cohesive ends. The oligolinkeris produced by annealing two oligonucleotides (with partialcomplementarity. The resulting plasmid is restricted with ClaI andEcoRV, which entails excision of the MORxx encoding sequence. Thesequence encoding the extracellular domain (ECD) of NPRA is amplifiedfrom the NPRA encoding plasmid (pcDNA3.1 DV5-His-TOPO-NPRA) by PCR usingprimers introducing restrictions sites for ClaI and SmaI, respectively.The PCR product is restricted with ClaI and SmaI, purified and thefragment was ligated into the above plasmid yielding a fusion of thekappa-leader, the extracellular domain of NPRA and human Fe. Theresulting plasmid is called pcDNA3.1_kappa-leader_NPRA-ECD_Fc.

B. Expression of the NPRA-Fc Fusion Protein

HEK293 cells are transfected with pcDNA3.1_kappa-leader_NPRA-ECD_Fcusing a calcium phosphate-based transfection procedure. On days 5 or 6post-transfection, the cell culture supernatant (1500 mL) is harvested,cleared by centrifugation and sterile filtrated (0.2 μm). Aliquots ofthe supernatant are frozen at −20° C.

Example 3 Methods Used for the Identification and Generation of HumanNPRA-Specific Antibodies from the HuCAL GOLD® Library

For the generation of therapeutic antibodies against the human NPRAprotein, selections with the MorphoSys HuCAL GOLD® phage display libraryare carried out. HuCAL GOLD® is a Fab library based on the HuCAL®concept [Knappik et al., J. Mol. Biol., 296, 57, 2000; Krebs et al., J.Immunol. Methods, 254, 67, 2001; Rauchenberger et al., J Biol Chem.,278, 38194, 2003], in which all six CDRs are diversified, and whichemploys the CysDisplay™ technology for linking Fab fragments to thephage surface [Löhning, WO 01/05950, (2001)].

A. Phagemid Rescue, Phage Amplification, and Purification

The HuCAL GOLD® library is amplified in 2×YT medium containing 34 μg/mlchloramphenicol and 1% glucose (2×YT-CG). After infection with VCSM13helper phages at an OD_(600nm) of 0.5 (30 min at 37° C. without shaking;30 min at 37° C. shaking at 250 rpm), cells are spun down (4120 g; 5min; 4° C.), resuspended in 2×YT/34 μg/ml chloramphenicol/50 μg/mlkanamycin/0.25 mM IPTG and grown overnight at 22° C. Phages arePEG-precipitated from the supernatant, resuspended in PBS/20% glyceroland stored at −80° C. Phage amplification between two panning rounds isconducted as follows: mid-log phase E. coli TG1 cells are infected witheluted phages and plated onto LB-agar supplemented with 1% of glucoseand 34 μg/ml of chloramphenicol (LB-CG plates). After overnightincubation at 30° C., the TG1 colonies are scraped off the agar platesand used to inoculate 2×YT-CG until an OD_(600nm) of 0.5 is reached andVCSM13 helper phages added for infection as described above.

B. Solid Phase Panning Against Captured NPRA-Fc with HuCAL GOLD®

This example describes solid phase panning which is used for selection.HuCAL GOLD® antibody-phage are divided into three pools corresponding todifferent VH master genes in combination with lambda and kappa lightchains (pool 1: VH1/5 lambda & kappa, pool 2: VH3 lambda & kappa, pool3: VH2/4/6 lambda & kappa). An additional pool (pool 4) is composed ofVH3 lambda & kappa of HuCAL GOLD® antibody-phage Hyperphage preparation.These pools are individually subjected to 3 rounds of solid phasepanning on NPRA-ECD-Fc captured on maxisorp plates (F96 Maxisorp, Nunc,Rochester, N.Y.) by an anti-Fc antibody (capture antibody). In detail:The wells of a maxisorp plate are coated with 100 μl of capture antibody(goat anti-human IgG Fc gamma Fragment specific, Dianova, HamburgGermany, 10 μg/ml in PBS). 3 wells per phage pool are coated. The plateis incubated overnight at 4° C. On the next day the wells are washedthree times with PBS and then blocked with 200 μl of MTBST (TBS, 0.05%Tween 20, 5% milk powder) for 2 h at RT. After washing three times withPBS, 100 μl of the NPRA-ECD-Fc containing cell supernatant are added andthe plate is incubated at RT for 4 h. Then the supernatant is discardedand 100 μl of fresh supernatant are added. The plate is stored overnightat 4° C.

The phage are arranged in 4 pools as described above. 100 μl of thephage from original HuCAL GOLD® subpools (VH1-6) each or of subpool VH3(HuCAL GOLD® Hyperphage preparation) are used, corresponding to1.7-8.0×10¹² phage. The phage are preblocked in a TBS solutioncontaining 2.5% milk powder, 0.05% Tween 20, 1% IgG Serum Goat(Dianova), 1% IgG Serum human (Dianova) and 2.5% FCS (PAN Biotech GmbH).The pre-blocking of phage is performed in 2 ml reaction tubes for 2 h atRT on a rotator.

For the selection process the NPRA-ECD-Fc supernatant is removed fromthe maxisorp plate and the wells are washed three times with PBS. Thepre-blocked phage are added to the corresponding wells and the plate isincubated for 2 h at RT on a microplate shaker. Then the phage solutionis removed and the wells are washed as follows: three times with PBST(PBS, 0.05% Tween 20), twice with PBST (with an incubation of 5 minbetween the washing steps), three times with PBS and finally twice withPBST (with an incubation of 5 min between washing steps). For elution ofspecifically bound phage 150 μl of 20 mM DTT in 10 mM Tris/HCl, pH 8.0is added and the samples are incubated for 10 min at RT. The eluates areused to infect log phase E. coli TG1 cultures. Infected E. coli areharvested by centrifugation and plated onto LB agar plates supplementedwith 34 μg/ml chloramphenicol and 1% glucose. The agar plates areincubated overnight at 30° C. On the following day the colonies arescraped off and grown until reaching an OD of 0.5 to proceed to helperphage infection.

Helper phage infection: TG1 cells are infected with the helper phageVCSM13 (multiplicity of infection of ˜20) at 37° C. The infected cellsare harvested by centrifugation and resuspended in 2×YT containing 34g/ml chloramphenicol, 50 μg/ml kanamycin and 0.25 mM IPTG for inductionof Fab expression. The cells are grown overnight and the produced phageare precipitated from the supernatant with polyethylene glycol(PEG)/NaCl and resuspended in PBS. Input and output titers aredetermined by spot titration.

Three rounds of selection are performed with increasing washingstringency. Between each round the eluted phage are precipitated asdescribed above.

C. Alternating Panning Using NPRA Expressing Cells

Selections can also are performed using whole cell panning with cellsthat express the receptor. For these selections, HuCAL GOLD®antibody-phage are divided into three pools as described above and anadditional pool (pool 4) is composed of VH3 lambda & kappa of HuCALGOLD® antibody-phage Hyperphage preparation. These pools areindividually subjected to one round of solid phase panning onNPRA-ECD-Fc, followed by one round on NPRA-expressing HEK cells,followed by another round of solid phase panning. The 1st round and 3rdround solid phase pannings are performed as described in section 3.4.1.The 2nd round panning is a whole cell panning on NPRA-expressing HEKcells followed by pH-elution.

More particularly, in the second round, all steps are carried out at 4°C. and in a volume of 1 ml in total. After detachment in Versene (GibcoInvitrogen, Carlsbad, Calif.) cells are washed twice in blocking buffer(5% FCS/0.05% NaN3/PBS) and adjusted to 1.0×10⁷ cells for each antibodyphage pool. The phage selected from the 1st round of the panning areincubated in blocking buffer for 2 h and then added to the pre-blockedcells for 2 h under constant movement. Afterwards, cells are washed fivetimes in blocking buffer followed by incubation in 1 ml elution buffer(0.1 M glycine, 0.5 mM NaCl, pH 2.2) for 10 min without shaking. Cellsare removed by centrifugation and the supernatant neutralized by theaddition of unbuffered 2 M Tris solution.

The eluate is mixed with a 15 ml culture of E. coli TG1 grown to anOD600 nm of 0.6-0.8 and incubated for 45 min at 37° C. Aftercentrifugation the bacterial pellet is resuspended in 2×YT medium,plated on 2×LB/Chloramphenicol/Glucose agar plates and incubatedovernight at 30° C. The selected clones are then scraped from theplates, rescued and amplified as described above.

D. Alternating Panning Against NPRA in the Presence of ANP and BNP

Selection can also be performed in the presence of the ligands for NPRA.For these selections, HuCAL GOLD® antibody-phage are not divided intothree pools, but all different VH master genes in combination withlambda and kappa light chains are mixed and subjected to one round ofsolid phase panning on NPRA-ECD-Fc, followed by one round onNPRA-expressing HEK cells, followed by another round of solid phasepanning.

Nevertheless two different pools are made, pool 1 consisting of HuCALGOLD® antibody phage normal preparation, pool 2 consisting of HuCALGOLD® antibody-phage Hyperphage preparation. The 1st round and 3rd roundsolid phase pannings are performed as described above with the followingexceptions:

The blocking solution for the phage contained 3 μM ANP and BNP each. Thecoating with the NPRA-Fc protein is much shorter; supernatant is onlyadded once, and the incubation is for 1 h. After this incubation thewells are washed three times with PBS, then 100 μl of ANP/BNP solution(3 μM each) is added and the plate is incubated 30 min at RT on amicroplate shaker. 8 wells per pool are coated for the selection.

The 2nd round panning is a whole cell panning on NPRA-expressing HEKcells followed by pH-elution as described above with the followingexceptions: The blocking solution for the phage contained 400 nM ANP andBNP each. After harvesting the NPRA-transfected HEK-cells areresuspended in blocking buffer containing 1 μM ANP. The mixture of cellsand ANP is incubated 30 min at 4° C. under constant movement beforeadding the blocked phage.

E. Subcloning and Expression of Soluble Fab Fragments

The Fab-encoding inserts of the selected HuCAL GOLD® phagemids aresub-cloned into the expression vector pMORPH®X9_Fab_FH or the bivalentexpression vector pMORPH®9_Fab_dHLX_MH respectively in order tofacilitate rapid and efficient expression of soluble Fabs. For thispurpose, the plasmid DNA of the selected clones is digested with XbaIand EcoRI, thereby excising the Fab-encoding insert (ompA-VLCL andphoA-Fd), and cloned into the XbaI/EcoRI-digested expression vectors.Fabs expressed from these vectors carry two C-terminal tags for both,detection and purification. In case of vector pMORPH®X9_Fab_FH the tagsare FLAG™ and 6×His; in case of pMORPH®X9_Fab_dHLX_MH the tags aremyc-tag and 6×His, respectively.

F. Microexpression of HuCAL GOLD® Fab Antibodies in E. coli

Chloramphenicol-resistant single colonies obtained after subcloning ofthe selected Fabs into the pMORPH®X9_Fab_dHLX_MH expression vector areused to inoculate the wells of a sterile 96-well microtiter platecontaining 100 μl 2×YT-CG medium per well and grown overnight at 37° C.5 μl of each E. coli TG-1 culture is transferred to a fresh, sterile96-well microtiter plate pre-filled with 100 μl 2×YT medium supplementedwith 34 μg/ml chloramphenicol and 0.1% glucose per well. The microtiterplates are incubated at 30° C. shaking at 400 rpm on a microplate shakeruntil the cultures are slightly turbid (˜2-4 hrs) with an OD_(600nm) of˜0.5.

To these expression plates, 20 μl 2×YT medium supplemented with 34 μg/mlchloramphenicol and 3 mM IPTG (isopropyl-β-D-thiogalactopyranoside) isadded per well (end concentration 0.5 mM IPTG), the microtiter platesare sealed with a gas-permeable tape, and incubated overnight at 30° C.shaking at 400 rpm.

Generation of whole cell lysates (BEL extracts): To each well of theexpression plates, 40 μl BEL buffer (2×BBS/EDTA: 24.7 g/l boric acid,18.7 g NaCl/l, 1.49 g EDTA/l, pH 8.0) containing 2.5 mg/ml lysozyme isadded and incubated for 1 hr at 22° C. on a microtiter plate shaker (400rpm). The BEL extracts are used for primary screening in the cGMP-assay.

G. Expression of HuCAL GOLD® Fab Antibodies in E. coli and Purification

Expression of Fab fragments encoded by pMORPH®X9_Fab_FH in TG-1 cellsare carried out in shaker flask cultures using 750 ml of 2×YT mediumsupplemented with 34 μg/ml chloramphenicol. Cultures are shaken at 30°C. until the OD_(600nm) reached 0.5. Expression is induced by additionof 0.75 mM IPTG for 20 h at 30° C. Cells are disrupted using lysozymeand Fab fragments isolated by Ni-NTA chromatography (Qiagen, Hilden,Germany). Protein concentrations is determined by UV-spectrophotometry[Krebs et al., J. Immunol. Methods, 254, 67-84 (2001)].

H. Cloning of HuCAL® IgG4

In order to express full length immunoglobulin (Ig), variable domainfragments of heavy (VH) and light chains (VL) are subcloned from thepMORPH®X9_FH Fab expression vectors into the pMORPH®_h_Ig vector seriesfor human IgG4.1Pro. Restriction enzymes EcoRI, MfeI, and BlpI are usedfor subcloning the VH domain fragment into pMORPH®_h_IgG4.1Pro: thevector backbone is generated by EcoRI/BlpI digestion and extraction ofthe 6400 bp fragment whereas the VH fragment (350 bp) is produced bydigestion with MfeI and BlpI and subsequent purification. Vector andinsert are ligated via compatible overhangs generated by the EcoRI andMfeI digests, respectively, and via the BlpI site. Thereby, both theEcoRI and the MfeI restriction site are destroyed. Subcloning of the VLdomain fragment into pMORPH®_h_Igκ is performed via the EcoRV and BsiWIsites, whereas subcloning into pMORPH®_h_Igλ is done using EcoRV andHpaI.

I. Transient Expression and Purification of Human IgG

Eukaryotic HKB11 or HEK293 cells are transfected with an equimolaramount of IgG heavy and light chain expression vector DNA. Cell culturesupernatant is harvested from 3 to 7 days post transfection. Afteradjusting the pH of the supernatant to 8.0 and sterile filtration, thesolution is subjected to standard protein A affinity chromatography(rProteinA FF or MabSelect SURE, GE Healthcare). Buffer exchange isperformed to 1×Dulbcecco's PBS (pH 7.2, Invitrogen) and samples aresterile filtered (0.2 μm). Purity of IgG is analyzed under denaturing,reducing conditions in SDS-PAGE or by using Agilent BioAnalyzer and innative state by SE-HPLC.

Example 4 Methods Used for the Screening of NPRA-Binding Fabs

A. Screening for NPRA Binding Fabs by ELISA

For some pannings ELISA is used as the primary screening method foridentifying for NPRA-Fc binding Fabs. NPRA-Fc is captured in microtiterplates by a goat anti-human IgG. A nonrelevant Fc fusion protein servesas a negative control to exclude Fabs, which are directed against thehuman Fc. Briefly, the capture antibody, Affinity Pure Goat anti humanIgG Fc-gamma specific (Dianova) at 10 μg/ml in PBS, is coated onMaxisorp® microtiter plates (Nunc) overnight at 4° C. On the followingday, the wells are blocked for 1 hour with MPBST (PBS/0.05% Tween 20/5%milk powder) on a microplate shaker. NPRA-ECD-Fc containing cellsupernatant is added and incubated for 1 hour at room temperature. Thewells of the microtiter plate are then washed three times with PBST(PBS/0.05% Tween 20). If required, a mixture of 150 nM ANP/300 nM BNP isadded and the plates are incubated and washed with PBST. Then HuCAL® Fabantibodies are added to the wells and incubated for 1 hour at roomtemperature. For detection of the primary antibodies, alkalinephosphatase (AP)-conjugated AffiniPure F(ab′)2 fragment, goat anti-humanIgG (Dianova, 109-055-097) is applied. For the development ofAP-conjugates, the fluorogenic substrate AttoPhos (Roche) is usedaccording to the instructions of the manufacturer. The plates are readin an ELISA-reader (Tecan).

B. Screening for NPRA Binding Fabs by FACS

HEK NPRA cells are detached with Accutase (PAA Laboratories GmbH, Cat.No. L11-007, Austria), harvested by centrifugation (900 rpm, 4 minutes)and resuspended in FACS buffer (PBS/3% FCS/0.02% NaN3) to a finalconcentration of 10⁶ cells/ml. 100 μl of the cell suspension istransferred to each well of a 96 round bottom plate (TC Microwell 96U,Nunc). Cells are pelleted (2000 rpm, 5 min, 4° C.), and resuspended in100 μl of FACS buffer with or without 100 nM ANP and incubated on icefor 30 min. Cells are washed in 150 μl of FACS buffer, pelleted andresuspended in 50 μl of the solution containing the primary antibodycomprising a BEL extract of Fabs, a diluted BEL extract or purifiedantibody diluted in FACS buffer and incubated on ice for 30 min. Cellsare washed again in FACS buffer and resuspended in 50 μl of FACS buffercontaining the secondary antibody (R-Phycoerythrin-conjugated AffipureF(ab′)₂ Fragment Goat Anti-Human IgG, Dianova) and incubated on ice for45 min. Cells are washed twice with FACS buffer and resuspended in 200μl of FACS buffer for analysis in the FACS Array (BD Biosciences).

C. Screening for Agonistic or Potentiating Fabs in the HEK NPRA cGMPAssay

The activation of NPRA results in generation of cGMP, the amount ofwhich is determined using the HitHunter™ cGMP Assay Kit (DiscoverX,Fremont, Calif.). The assay buffer used is PBS/0.1% BSA/25 mM HEPEScontaining 1 mM of the phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX, Sigma). The assay is performedaccording to the protocol. For a standard curve 10-fold dilutions ofcGMP between 4 pM and 4 μM are used. Fabs are screened in thedHLX-format as BEL lysates, micropurified Fabs or large scalepurifications. The screening is performed in presence or absence of asuboptimal concentration of ANP (final concentration in assay 40 pM),which does not elicit a cGMP response on its own.

For the screening assay, ANP is diluted in assay buffer to aconcentration of 160 pM and 7.5 μl of this solution was pipetted intothe wells of a 96 round bottom well plate (TC Microwell 96U, Nunc). Then7.5 μl of Fab is added. NPRA-transfected HEK cells are harvested, washedand resuspended in assay buffer to a final concentration of 3.3×10⁵cells per ml. 15 μl of the cell suspension is added to the wells and theplate is incubated for 15 min at 37° C. Then the cells are lysed byadding 20 μl of lysis buffer and anti cGMP antibody reagent (mixed 1:1).Immediately afterwards 20 μl of ED reagent is added and the plate isincubated for 1 h at room temperature. Then 20 μl of EA reagent is addedand the plate is incubated for another 30 min. During that incubationthe samples are transferred to a plate suitable for determination ofluminescence (OptiPlate-96, Perkin Elmer). Then 30 μl of substratesolution (Galacton Star:Emerald II:Substrate Diluent 1:5:19) is added.Luminescence is measured in a TECAN reader after 2 to 3 h.

Example 5 Identification of a Human NPRA-Specific Antibody

A. Initial Pannings.

The initial two pannings are whole cell pannings performed onNPRA-transfected HEK-cells with postadsorption on untransfectedHEK-cells. One of the pannings is performed with conventional acidicelution, in the other panning the NPRA ligands ANP and BNP are added inorder to displace the potential binding Fab on the antigen and thuselute the corresponding phage. From each panning 1140 candidates arescreened in ELISA for NPRA-ECD-Fc binding and 276 candidates arescreened in FACS for specific binding to NPRA-transfected cells. Nospecific Fabs are found in either screen.

Solid phase pannings are performed on immobilized NPRA-ECD-Fc and analternating panning against NPRA. The Fabs are subcloned into thepMx9_FH vector. The primary screening is performed via ELISA againstNPRA-ECD-Fc. Out of 1532 Fabs tested, 123 show specific binding toNPRA-ECD. Fabs that bind to Fc or to the capture antibody are excludedafter detection by an ELISA against a captured control Fc-protein. Thesecondary screening consists of a FACS scan looking for binding toNPRA-transfected HEK-cells.

Out of 123 primary hits 49 show at least weak binding (threshold 2-foldover background). These 49 clones are sequenced and 6 unique sequenceswere identified. However, these Fabs do not show agonistic orpotentiating activity in the cGMP assay, either in the absence of ligandor in presence of suboptimal concentrations of ANP.

With the intention of generating antibodies against the activeconformation of the NPRA receptor, the activating ligands ANP and BNPare added during the a series of solid phase pannings on NPRA-ECD-Fc aswell as on an alternating panning on NPRA expressing cells.

The selected Fab pool is subcloned into the pMx9_dHLX_MH vector. Theprimary screening is done via ELISA against NPRA-ECD-Fc in presence ofANP. Out of 1528 Fabs tested, 299 show specific binding to NPRA-ECD inpresence of ANP. Fabs that bind to Fc-protein or the capture antibodyare detected by an ELISA against a captured control Fc-protein andexcluded. 177 of the 198 hits derived from the alternating panning arealso tested for binding to NPRA-transfected HEK cells in FACS. 175 ofthese show specific binding. The secondary screening is performed viacGMP assay performed in presence of suboptimal concentration of ANP (40pM) in order to allow for the detection of Fabs which would not elicitcGMP response on their own, but would elevate ANP-elicited response.

ANP at 40 pM on its own does not stimulate a detectable cGMP response inthe assay. BEL extracts of the Fabs were used. Of the 299 ELISA hits 279are tested in the cGMP assay. None of them show significant agonisticactivity, but 69 Fabs which showed a slightly elevated activity arechosen for further investigation after micropurification.Micropurification is preferred, because it yields a higherFab-concentration and because some tests show that BEL-buffer inhibitsthe assay to some extent. Of the 69 Fabs 9 are chosen for furtherinvestigation based on elevated cGMP response. For 4 out of 9significant elevation of cGMP levels are confirmed. These clones aresequenced and proven to be identical. The Fab identified is given thename 5064.

B. Characterization of HuCal Gold Selected Fabs.

Several non-agonistic Fabs against NPRA when selected and converted tothe IgG1 format bind to HEK hNPRA cells with EC50s between 10 and 20 nMwith different saturation levels. However, none of these binders, in amonovalent (Fab) or bivalent (Fab-dHLX) format, display agonistic orpotentiating activity in absence or presence of suboptimalconcentrations of ANP. These antibodies are not investigated further.

In contrast, the binder 5064, in both the monovalent (Fab) and bivalent(Fab-dHLX) formats show specific and concentration dependent binding toNPRA transfected HEK293 cells only in the presence of ANP or BNP, i.e.this binder specifically recognizes the receptor-ligand complex (FIG.1). Fab-dHLX shows stronger binding than the Fab indicating increasedavidity due to bivalency. Both monovalent and bivalent 5064 revealstronger binding to the ANP bound receptor compared to BNP boundreceptor, which may be related to the higher affinity of ANP for NPRA.Titration of 5064 Fab, Fab-dHLX and IgG1 over a broader concentrationrange allows an estimation of EC50 values for cell binding. 5064 Fabdisplays an EC50 of at least ˜100 nM and ˜300 nM on ANP and BNP boundHEK NPRA cells, respectively. The corresponding bivalent antibodyformats, Fab-dHLX and IgG1, exhibited about 50 to 100-fold strongerbinding than the Fab (FIG. 2A-2C).

To further assess the specificity of the binder 5064, binding studieswith recombinant NPRA-Fc are performed. As previously observed withcellular NPRA, the antibody is able to bind NPRA-Fc only when it wasloaded with the ligands ANP or BNP (FIG. 3). Since it may be possiblethat 5064 binds only to the peptide ligands and not to the receptor,binding of the antibody to the free ligand is investigated. To this endbiotin-labeled ANP is conjugated to Streptavidin beads and binding ofantibodies is determined. While a positive control antibody against ANPshows significant binding, 5064 Fab-dHLX shows absolutely no binding(FIG. 4). In another experiment 5064 Fab is pre-incubated with an excessof ANP in solution before being added to HEK NPRA cells loaded with ANP(FIG. 5). In this setting competition with excess ANP has no effect onbinding of 5064 to the ANP-NPRA complex. Taken together, these findingsshow that 5064 specifically recognizes the activated ligand-receptorcomplex, but does not interact with either receptor or natriureticpeptides alone.

The effects of 5064 on the guanylyl cyclase activity of NPRA isevaluated by incubating the binder in the Fab-dHLX format with HEK NPRAcells in the absence or presence of 40 pM ANP and measuring NPRAdependent production of cGMP. In the absence of ANP, the cGMP responsewas not increased above baseline at concentrations of 5064 up to 100μl/ml (˜900 nM). In presence of 40 pM ANP a minimal cGMP signal isobserved with or without negative control Fab-dHLX. However in presenceof 40 pM ANP and 5064 Fab-dHLX at concentrations above 50 μg/ml (˜450nM) the cGMP level is significantly elevated (FIG. 6). A similar effectis observed when 5064 Fab, Fab-dHLX and IgG1 are tested at variousconcentrations in presence of 40 nM BNP, which is insufficient to inducecGMP on its own (FIG. 7). 5064 IgG enhances the NPRA dependent cGMPproduction at concentrations between 10 and 100 nM, it was followed byFab-dHLX, which increases the cGMP signal at concentrations above 100nM. The monovalent Fab showed the weakest effect, which could only beobserved at the highest concentration of 400 nM.

To further assess the effects of 5064 on the potency of ANP and BNP inthe activation of NPRA, 20 μg/ml of 5064 or a negative control Fab orFab-dHLX is added to HEK NPRA cells in the presence of increasingconcentrations of ANP or BNP and cGMP production is measured. In thepresence of either 5064 Fab or Fab-dHLX the potency of ANP and BNP isincreased by 2 to 3-fold as seen by a shift of the dose-response curvesto the left (FIG. 8A and FIG. 8B). 5064 potentiates ANP and BNP-mediatedNPRA activation only at submaximal concentrations of natriureticpeptides. No further increase in cGMP production is observed atsaturating ligand levels. 5064 uniquely enhances the NPRA dependent cGMPresponse to natriuretic peptides. Other HuCAL® antibodies that areselected for their ability to bind to NPRA either have no effect on cGMPproduction or inhibit it (FIG. 10).

Example 6 Optimization of the Anti-NPRA Binder 5064 Through AffinityMaturation

Altogether these data support the hypothesis that 5064 is able to bindand stabilize the activated NPRA receptor and thereby enhance theactivity of natriuretic peptides. However, 5064 is the only binder,which shows this activity. Since this binder only has moderate affinityin the monovalent format (EC50 approx. 100 nM), the affinity of thisbinder is further optimized in order to potentially increase itsbiological activity.

A. Generation of Affinity Maturation Libraries

To increase the affinity and biological activity of the anti-NPRA 5064Fab, L-CDR3 and HCDR2 regions are optimized in parallel by cassettemutagenesis using trinucleotide directed mutagenesis (Virnekas et al,1994), while the framework regions are kept constant. Prior to cloningfor affinity maturation, the parental Fab fragment is transferred fromthe corresponding expression vector (pMORPH®x9_FH) into the CysDisplay™vector pMORPH®25_LHC via XbaI/EcoRI. pMORPH®25_LHC is created from theHuCAL GOLD® display vector pMORPH®23_LHC by removal of one BssHII siteinterfering with library cloning for H-CDR2 optimization. For optimizingL-CDR3 the L-CDR3 and the constant region of the light chains of theparental Fab are removed by BbsI/SphI and replaced by a repertoire ofdiversified L-CDR3s together with the constant domain. In a secondlibrary the H-CDR2 (XhoI/BssHII) is diversified, while the connectingframework regions are kept constant. In order to monitor the cloningefficiency the parental H-CDR2 is replaced by a dummy, before thediversified H-CDR2 cassette is cloned in. Ligation mixtures of the twodifferent libraries are electroporated in E. coli TOP10F cells(Invitrogen, Carlsbad, Calif., USA) yielding from 5×10⁸ to 8×10⁸independent colonies. This library size ensured coverage of thetheoretical diversity. Amplification of the library is performed asdescribed before (Rauchenberger et al., 2003). For quality controlsingle clones are randomly picked and sequenced (SequiServe,Vaterstetten, Germany).

B. Whole Cell Panning Against NPR4A-HEK Cells

The phage derived from the above maturation libraries (HCDR2 and LCDR3maturation respectively) are individually subjected to 3 rounds of wholecell panning on NPRA-transected HEK-cells. Three different conditionsare applied: Condition 1 is a whole cell panning in presence of ANP/BNP.Condition 2 is a whole cell panning in presence of ANP/BNP withcompetition by parental binder 5064. Condition 3 is a whole cell panningin absence of ANP/BNP. Thus 6 whole cell pannings are performed.

All steps are carried out at 4° C. After detachment with Accutase (PAALaboratories, L11-007) cells are counted, harvested and adjusted to5×10⁶ cells per panning for the 1^(st) round. For the 2nd round 1×10⁶cells per panning are used and for 3rd round 5×10⁵ cells per panning.The cells are resuspended in 1.5 ml of blocking buffer (PBS/5% FCS/0.05%NaN₃) and incubated for 30 min on a rotator. For panning conditions 1and 2 ANP and BNP are added to a final concentration of 100 nM each inthis incubation step. The cells are harvested by centrifugation (2 min,2000 rpm) and resuspended carefully in the solution containing thepre-blocked phage. Before that step, 83 μl of phage (corresponding to5.1×10⁵ for HCDR2 matured library and 9.8×10¹¹ for LCDR3 maturedlibrary) per panning has been pre-blocked by mixing with 917 μl ofblocking buffer and incubated for 2 h on a rotator. During theincubation of cells with pre-blocked phage ANP and BNP are added to afinal concentration of 100 nM each for conditions 1 and 2.

For condition 2 the parental Fab 5064 is added to a final concentrationof 200 nM, but only after cells and phage had already been incubated for1.5 h. For all conditions the total time of incubation is 2 h. Thencells are harvested by centrifugation (2 min, 2000 rpm) andnonspecifically bound phage are washed off by incubation with 1.5 ml ofblocking buffer on a rotator. The washing steps are performed asfollows: 5×10 min in the 1st round, 5×20 min in the 2nd round and 6×20min in the 3rd round. For condition 2 parental Fab 5064 is added to afinal concentration of 200 nM in each washing step. Specifically boundphage are eluted from cells and subsequent steps are carried out asdescribed above.

C. Panning Against NPRA-Fc Captured on Beads

Three different conditions are applied: Condition 1 and 2 are panningsin presence of 30 nM ANP and BNP each. Condition 3 is a panning inabsence of ligands. The antigen concentration is varied depending on thepanning condition by using a different amount of NPRA-Fc-coated beads orby using different dilutions of the NPRA-ECD-Fc containing cellsupernatant. All plastic tubes used are pre-blocked by filling with 1.5ml of PBS/5% BSA and incubating them overnight at 4° C. on a rotator.

The NPRA-Fc protein is captured on Dynabeads® (electromagnetic M-280Streptavidin beads, 10 mg/ml, Dynal) via a biotinylated anti-Fc antibody(mouse anti-human Fc, Chemicon, #CBL 102, biotinylated in PC-group).Between incubation steps the beads are washed with 1.5 ml of By buffer(PBS/0.05% BSA/0.02% Tween) and then harvested using a magnet.

The beads are prepared as follows: 60 μl of beads are mixed with 1437 μlof Bv-buffer and with 2.05 μl of biotinylated CBL 102 and the mixture isincubated for 30 min at 22° C. on a rotator. The beads are washed, 1.5ml of the NPRA-ECD-Fc containing cell supernatant is added and themixture is incubated for 90 min at 22° C. on a rotator. Then the beadsare washed again, resuspended in 1 ml of By buffer and then split into 2fresh tubes, each containing 0.5 ml of the NPRA-Fc coated beads insuspension. In one tube ANP and BNP are added to a final concentrationof 30 nM each and both tubes are incubated for 30 min at 22° C. on arotator. Both samples are washed again in By buffer and resuspended in0.75 ml of By buffer. Then the beads are ready to use. The beads arecoated fresh for each round of the panning.

Simultaneously to coating the beads, 83 μl of phage (corresponding to5.1×10¹¹ for HCDR2 matured library and 9.8×10¹¹ for LCDR3 maturedlibrary) per panning are pre-blocked by mixing with 5 μl of Ig Serummouse (Mouse Gamma Globulin, Dianova, 015-000-002) and 712 μl ofPBS/0.05% Tween/5% BSA and incubating for 2 h on a rotator.

The beads treated with ANP and BNP are used for panning condition 1 and2. The beads without ligands are used for panning condition 3. For the1st round 200 μl of the beads “ready to use” preparation are used.

For selection, 200 μl of beads (1st round) are mixed with 800 μl ofblocked phage and incubated for 2 h on a rotator. The washing steps areas follows: 5× quick wash in PBS/0.05% Tween, 3×15 min in PBS/0.05%Tween on a rotator, 4× quick in PBS and 3×5 min in PBS on a rotator.After washing the beads are transferred into a fresh tube.

For elution, beads are harvested and resuspended in 300 μl of 20 mM DTTin 10 mM Tris/HCl, pH 8.0 and incubated for 10 min. Then the beads areharvested and the phage containing supernatant is used to infect E. coliTG1 as described above.

In order to identify Fab clones with improved binding to cell-bound NPRAafter affinity maturation, a modified FACS screening procedure is used:BEL extracts (whole cell lysates) of Fab-expressing bacterial clones arescreened for specific cell binding via FACS. BEL extracts are diluted1:50 in FACS buffer and used for FACS screening according to standardprotocol. The dilution is chosen, because at that concentration bindingof the parental Fab is barely detectable. Clones displaying FACS signalssignificantly above values obtained for the parental controls are pickedfor further characterization.

D. Characterization of Affinity Matured Fabs

From both pannings on cells and NPRA-Fc protein more than one hundredhits are identified and 94 of these binders are micro-purified andfurther analyzed in cell binding and cGMP assays. Out of the 94 Fabs, 21show superior performance in cell binding and/or the cGMP assay.Sequencing of these 21 binders reveals 14 unique clones, which arepurified.

Three of the 14 Fabs are derived from the cell pannings and 11 areselected in pannings on NPRA-Fc. Nine Fabs are H-CDR2 matured and fiveFabs are L-CDR3 optimized. All of them are derived from pannings done inpresence of ANP.

Fourteen selected Fabs purified in mg-scale are analyzed in cell bindingand cGMP assays. The Fabs are tested at various concentrations forbinding to HEK NPRA cells loaded with 100 nM ANP (FIG. 10A). In thisstudy Fab 5504 displays the strongest binding followed by Fabs 5502,5507, 5513, 5514 with slightly weaker affinities but still in the lownanomolar range. Interestingly, the five L-CDR3 matured Fabs show thestrongest binding to human NPRA transfected cells, whereas the H-CDR2matured Fabs in most cases display weaker binding.

The potentiating activity of the Fabs is assessed by measuring cGMPproduction in HEK NPRA cells in the presence of a suboptimalconcentration (400 pM) of ANP (FIG. 11). This ligand concentration issufficient to induce a significant but not a full cGMP response on itsown. As compared to the negative control Fab 3207, the parental Fab 5064displays a slightly increased cGMP signal. At least some of the maturedFabs (5502, 5504, 5507, 5511, 5513, 5514) appear to induce higher cGMPlevels than 5064 under these experimental conditions. Based on cellbinding data and the analysis of the cGMP response the L-CDR3 maturedFabs 5502, 5504, 5507, 5513, 5514 and the H-CDR2 matured Fab 5511 areanalyzed in more detail.

In this study increasing concentrations of ANP is added to HEK NPRAcells in the presence of 400 nM of the selected Fabs and cGMP productionis monitored (FIG. 12A). As seen previously the parental Fab 5064decreases the EC50 of ANP from 3 nM (in presence of negative controlFab) to 1 nM. Two of the matured Fabs, 5502 and 5504, are able to shiftthe EC50 of ANP further down to 0.3 nM and thus enhance the potency ofANP by a factor of 10. Three other matured Fabs, 5507, 5513, 5514,display intermediate activities resulting in EC50 values of 0.5-0.6 nMfor ANP. The impact of the Fabs on the dose-response of BNP is alsoanalyzed. Although the effect is less pronounced as for ANP, the maturedFabs clearly enhance the potency of BNP. While the parental Fabdecreases the EC50 of BNP only 2-fold, the matured Fabs show a 5-folddecrease down to 18 nM for Fab 5504 compared with an EC50 of 90 nM inpresence of negative control Fab (FIG. 12B).

Altogether, the data provide evidence that affinity maturation of 5064results in elevated binding of the resulting Fabs to the NPRA-ligandcomplex as well as in increased potentiation of ANP and BNP dependentcGMP production in NPRA overexpressing cells.

The binders 5502, 5503, 5504, 5507, 5508, 5511, 5513, 5514 are convertedinto the IgG4-Pro format. The IgG4 subtype is chosen to minimizeeffector function of the resulting antibodies and the proline 228 toserine mutation is introduced to abrogate Fab arm exchange (van der NeutKolfschoten, et al Science 317:1554, 2007). The most promisingcandidates are those that have alterations in the light chain (5502,5504, 5507, 5513, 5514). The light chains of these IgGs are crosscombined with the heavy chain of matured IgG 5511, because the 5511 Fabhad shown some increased activity compared to the parental 5064 in cGMPassay (FIG. 11).

The affinity matured and cross combined IgG4_Pro antibodies are analyzedat various concentrations by FACS for binding to HEK_NPRA loaded with100 nM ANP (FIG. 13A and FIG. 13B). While the parental IgG4_Pro 5064shows half-maximal binding at a concentration of 1.6 nM, the maturedantibodies displayed increased binding with EC50 values down to 0.5 nM,most likely at the sensitivity limit of the assay.

The selected IgG4_Pro antibodies are also analyzed for their capacity toincrease the potency of ANP and BNP in the cGMP assay. A fixedconcentration of 200 nM of antibody is applied with variousconcentrations of ANP or BNP to HEK NPRA cells. FIGS. 14A and 14B and15A and 15B show representative cGMP assays. The matured antibodiesfurther potentiate ANP and BNP dependent cGMP production as compared tothe parental antibody 5064. 5502, 5504, 5591 and 5592 induce the largestshift with up to a 10-fold increase in the potency of ANP.

To determine whether the profiled antibodies are selective for NPRA,binding to the related natriuretic peptide receptors NPRB and NPRC isassessed by FACS. While the matured (FIG. 16A) and cross combined (FIG.16B) antibodies bind to HEK NPRA cells incubated with ANP, no binding isobserved to HEK NPRB cells in the presence or absence of its ligand CNP.Similarly no binding is seen to HEK NPRC cells in the presence orabsence of ANP or BNP (FIG. 17).

Example 7 Determination of Antibody Binding Affinity to NPRA—NatriureticPeptide Complexes

A. Binding Affinity Determination by Quantitative FACS Analysis

The binding affinity of selected antibodies to NPRA complexed to itpeptide ligands is quantitated utilizing several different technologies.In one assay, the binding to HEK NPRA cells in the presence of 100 nMANP is evaluated by FACS analysis as outlined above. The parentalIgG4_Pro 5064 shows half-maximal binding at a concentration of 1.6 nMwhile the matured and cross combined antibodies display increasedbinding with EC₅₀ values in the nM range. However these values arethought to be at the limits of this assay.

TABLE 1 FACS analysis of the binding of IgG4_Pro antibodies to HEK NPRAcells in the presence of 100 nM AMP Antibody IgG4_Pro EC₅₀ (nM) 50641.08 ± 0.13 5502 0.68 ± 0.26 5504 0.56 ± 0.15 5591 0.73 ± 0.19 5592 0.52± 0.04

B. Binding Affinity Determination by Surface Plasmon Resonance (Biacore)

In another assay, the purified human NPRA-Fc fusion protein construct isused to develop a surface plasmon resonance (BIAcore) assay fordetermining affinity and kinetic constants for the antibodies. NPRA-Fcin 10 mM sodium acetate buffer, pH 5.5 is immobilized on a CM5 chip in aBiacore 3000 instrument (GE Healthcare, Biacore, Inc.) through aminecoupling at a density of 1000 RU. Increasing concentrations of anti-NPRAantibodies ranging from 0.5 to 7 nM in 10 mM HEPES, pH 7.4, 150 mM NaClsupplemented with 100 nM human ANP, BNP or urodilatin is injected in theflow cell at a flow rate of 20 μl/min for 10 minutes followed by a 15minute dissociation period. The collected association and dissociationdata from each experiment is globally fitted with the association(k_(a)) and dissociation (k_(d)) rates fit simultaneously. Summarized inthe tables below, the kinetic binding data demonstrate that theinteractions are of high affinity, in the range of 10 pM˜200 pM and thatthe antibodies bind to NPRA in the presence of all three of its naturalligands. The candidates have higher affinity in the presence of ANP thanin the presence of BNP and urodilatin. Anti-NPRA 5592 has higheraffinity than 5502 and 5592 in the presence of ANP, BNP or urodilatin.The data fit to a 1:1 binding model which suggests that one bivalentantibody binds to a single dimeric NPRA extracellular domain. 5591 and5592 Fabs bind to NPRA-Fc in the presence of ANP with lower affinity andalso with a 1:1 stoichiometry.

TABLE 2A Affinity and kinetic data of surface plasmon resonance analysisof interaction between Fabs or IgGs with NPRA-Fc in the presence of ANPFabs or IgGs K_(D) (pM) k_(a) (1/Ms) k_(d) (1/s) 5591 Fab 1160 8.84e51.03e−3 5592 Fab 426 868e5 3.69e−4 5502 IgG 172 1.65e6 2.83e−4 5591 IgG27.5 3.88e6 1.07e−4 5592 IgG 11.0 9.59e6 1.06e−4

TABLE 2B Affinity and kinetic data of surface plasmon resonance analysisof interaction between IgGs and NPRA-Fc in the presence of BNP IgGsK_(D) (pM) k_(a) (1/Ms) k_(d) (1/s) 5502 413 1.36e6 5.63e−4 5591 3802.69e6 1.02e−3 5592 45.9 7.42e6 3.40e−4

TABLE 2C Affinity and kinetic data of surface plasmon resonance analysisof interacton between IgGs and NPRA-Fc in the presence of urodilatinIgGs K_(D) (pM) k_(a) (1/Ms) k_(d) (1/s) 5502 211 2.12e6 4.48e−4 559192.8 2.56e6 2.38e−4 5592 25.1 3.96e6 9.94e−5

C. Binding Affinity Determination by KinExA

To confirm the anti-NPRA antibody affinity to cell-associated fulllength NPRA, a whole cell binding assay is done employing the KinExAtechnology. HEK NPRA cells ranging in concentration from 2×10⁷ cells/mlto 9.7×10³ cells/ml in cold Hank's Balanced Salt Solution with 0.5%bovine serum albumin are incubated in the presence of 1 nM ANP andeither 75 or 200 pM 5591 IgG for 1 hour at 4° C. The cells are pelletedand the supernatants are loaded into the KinExA instrument (SapidyneInstruments) where free IgG is captured on beads using mouse anti-humankappa light chain (Southern Biotech) and quantitated using goatanti-human IgG couple with Alexa Fluor 647 (Invitrogen). Using thismethodology, the apparent Kd for 5591 antibody binding is determined tobe 590 pM (FIG. 18). This value is about 20-fold higher that the K_(d)obtained though surface plasmon resonance binding to the NPRAextracellular domain-Fe fusion protein.

Example 8 Antibody Potentiation of NPRA Mediated cGMP Responses

The binding of peptide ligand to NPRA results in the activation of theguanylyl cyclase activity associated with this receptor. The effects ofthe antibodies on the production of cGMP in response to ligand aremeasured in HEK NPRA cells in suspension. The cells are incubated for 15minutes in the presence of 10 μg/ml of the NPRA specific IgG_Proantibodies or the control 3207 antibody, and increasing concentrationsof ANP or BNP. cGMP levels are quantitated using a competitiveimmunoassay with a luminescent readout (HitHunter kit, DiscoverX,Fremont, Calif.)). cGMP production dose response curves are generatedwith a four parameter logistic equation fitted using the LevenburgMarquardt algorithm in XLfit 4.2 data analysis software (ID BusinessSolutions, Ltd., Guildford, UK) and EC₅₀s are calculated. Fold shifts inan EC₅₀ represents the ratio between the EC50 generated in the presenceof an anti-NPRA antibody and that generated in the presence of an equalconcentration of the control antibody 3207.

5502, 5591 and 5592 reproducibly shift the ANP or BNP dose responsecurves to the left as illustrated in FIGS. 14 and 15. The magnitude ofthe shifts are enumerated in Table 3 and range from a maximum of 7-foldfor ANP to 44-fold for BNP. The ranges are reflective of individualexperiments with at least 3 experiments done under each condition. Theincreased fold shifts observed in the presence of the antibodies uponactivation of NPRA with BNP presumably results from the weaker affinityof this NPRA peptide ligand.

TABLE 3 Potentiation of Antibody mediated cGMP responses in ANP or BNPtreated HEK NPRA cells IgGs Shift in ANP EC₅₀ Shift in BNP EC₅₀ 5502 5-7fold 15-19 fold 5591 3-6 fold 15-16 fold 5597 3-5 fold 31-44 fold

The effects of the antibodies are most notable at the sub-optimal levelsof natriuretic peptide which, being in the low pM range, are consistentwith the concentrations of ANP and BNP observed in patients with heartfailure. Shifts of similar magnitude are seen in ANP treated HeLa cellswhich express NPRA endogenously.

In order to measure the biological potency of the antibodies, thesereagents are incubated with HEK NPRA cells in the presence of asub-optimal concentration of ANP (200 pm) and cGMP production ismonitored. The antibodies are dose responsive with EC₅₀ values around 1nM and about 10 fold more potent than the parental antibody 5064 (Table4).

TABLE 4 Titration of the anti-NPRA antibodies on HEK NPRA cells in thepresence of 200 pM ANP IgGs cGMP EC₅₀ (nM) 5064 12.1 5502 2.1 5504 1.25591 0.5 5592 1.0

Example 9 Prolongation of NPRA-Mediated cGMP Responses by 5591

In addition to enhancing activation of the receptor at low ligandconcentrations, the anti-NPRA antibodies also appear to extend thetimeframe of receptor signaling. This is demonstrated by monitoring thekinetics of NPRA-dependent cGMP production in HEK NPRA cells (FIG. 19).The cells are incubated with either a sub-optimal (200 pM) or an excess(1 pM) concentration of ANP in the absence or presence of 10 μg/ml 5591and cGMP production is measured over time as detailed above.

At sub-optimal levels of ANP, 5591 progressively enhances cGMPproduction over the 2 hour time frame of this experiment. In thepresence of a high concentration of ANP, where it would be predictedthat the receptor would already be maximally activated, the antibody hasno effect.

Example 10 Antibody Binding Stabilizes the Receptor Ligand Complex

In order to directly monitor the effects of the antibodies on theinteraction of a natriuretic peptide with its receptor, the binding ofradiolabeled ANP to HEK NPRA cells is examined. 100 μl of HEK NPRA cellsat 2×10⁶ cells/ml in DMEM (Invitrogen-Gibco) with 0.1% bovine serumalbumin (Sigma-Aldrich) are added to each well of Multiscreen HTS FC 1.2mm G1 96 well plates (Millipore Corporation, Billerica, Mass.) coatedwith 0.2% polyethylenimine (Sigma-Aldrich). 5502, 5504, the parentalantibody 5064 or the control antibody 3207 is added at a finalconcentration of 10 μg/ml. 100 μl/well of various concentrations of ¹²⁵IANP (GE Healthcare Bio-Sciences, Piscataway, N.J.) is then added to theappropriate wells and the plates are incubated at room temperature on anorbital shaker for two hours. The cells are washed four times with 200μl/well of cold DMEM with 0.1% BSA using a vacuum manifold, followed bya single wash with PBS supplemented with 25 mM HEPES, and 0.1% BSA. Theplates are air dried overnight, the bottom each plate is sealed with anopaque white plate sealer, and 30 μL of Microscint 40 is added to eachwell. The top of the plates are sealed with TopCount Plate Sealers andthey are read in a TopCount microplate scintillation counter (PerkinElmer). Non-specific binding is assessed by incubating the cells with¹²⁵I ANP in the presence of a 200 fold excess of cold ANP.

Initial studies reveal that this class of antibodies significantlyenhanced the binding of ¹²⁵I-ANP to HEK NPRA cells as compared to thecontrol antibody 3207 (FIG. 20). The enhanced binding is evident even atconcentrations of ANP below 200 pM (within the range of ANP levelsobserved in heart failure patients). One possible explanation for theobserved increased ¹²⁵I-ANP binding seen is that the antibodies slow therelease of ANP from the receptor. In order to test this hypothesis,1×10⁶ HEK NPRA cells/ml are incubated with 100 nM ¹²⁵I-ANP in thepresence of 10 μg/ml anti-NPRA or control antibody for 2 hours at 4° C.to form complexes. A 2000 fold excess of cold ANP is then added, and 100μl samples are transferred to wells in a filter plate over time and cellassociated radioactivity is quantitated as outlined above. In thepresence of the control antibody 3207 the off-rate of ANP is so rapidthat it is difficult to capture even at the zero time point (FIG. 21).However 5502, 5504, 5591 and 5592, significantly slow the release of ANPfrom the receptor. The parental antibody 5064 is less effective than thematured and cross-combined antibodies. These data are consistent with amodel in which the antibody binds to and stabilizes the ligand-boundconfirmation of the NPRA and sustains and enhances signaling though thisreceptor.

Example 11 Epitope Mapping by Hydrogen/Deuterium Mass Spectrometry(HXMS)

In order to identify the antibody binding site on the extracellulardomain of NPRA the technique of hydrogenideuterium mass spectrometry isutilized. This method is employed to study the solvent accessibility ofthe backbone amide hydrogens in a polypeptide and changes that occurupon antibody binding. It involves the exposure of NPRA-Fc, NPRA-Fc+ANPor NPRA-Fc+ANP+5591 IgG4_Pro to heavy water for various lengths of time.The hydrogen/deuterium exchange is quenched by low pH and temperatureand the protein complexes are rapidly degraded by pepsin and thenanalyzed by LC/MS to determine the extent of deuterium incorporationinto each peptide.

A. Deuterium Exchange Experiments—Identification of Peptides.

Peptides generated from NPRA-Fc by peptic digestion are identified. Theprotein is digested with pepsin, on ice, for 5 minutes at 0° C. Theresulting peptides are subjected to μHPLC/FTMS analysis. Mobile phase A(99/1/0.1, H₂O/Acetonitrile/Formic Acid) and Mobile Phase B (95/5/0.1,H₂O/Acetonitrile/Formic Acid). Flow rate is 100 μl min, with a 10 μlinjection onto a 150×1 mm HypersilC18 column. (Thermo PN 22105-396). Thegradient program is from 0% B to 100% B at 15 minutes, hold to 19minutes. FTMS spectra (Bruker Apex II, Billerica, Mass.) are acquiredfrom m/z 400-1800. Four 0.35 sec spectra are accumulated for each storedspectrum with a 256 K length, 50,000 resolution. External calibration isperformed by in source fragmentation of porcine renin substrate. Asecond run is made under identical conditions with the exception ofincreasing the capillary exit voltage of the interface from 80 V to 180V to produce fragment ions from the eluting peptides and alteration ofthe m/z range to 200-2000 and using 512K word acquisition. Data areconverted to MassLynx (Micromass/Waters, Manchester, UK) format foranalysis. All spectra are averaged into a single spectrum. This issubjected to analysis by MassLynx MaxENT3 to produce a list ofmonoisotopic molecular weights for each deconvoluted component. Thislist is searched versus the protein sequence to determine possiblepeptides without any constraints on the cleavage sites with the use of a5 ppm error window. The corresponding spectrum is extracted from thehigh capillary exit voltage run. The fragment ions present in thespectrum are used for comparison to theoretical fragmentation patternsgenerated by the MassLynx software.

B. Hydrogen/Deuterium Exchange Experiments

NPRA-Fc, NPRA-Fc+ANP or NPRA-Fc, +ANP+5591-IgG complexes are diluted1:10 into deuterated 5 mM NaH₂PO₄ buffer at pH 7.0 and allowed toexchange for varying lengths of time (5, 15, 45, 100, 1000 seconds) atroom temperature. Exchange is quenched by lowering the pH andtemperature (adding an equal volume of ice-cold 100 mM NaH₂PO₄, pH 2.5).The protein is then digested with pepsin (1:1 for 5 minutes on ice) andinjected onto a trap column (Michrom C18) and then a 0.8×150 mm C18(PepMap C18, LC Packings) column for separation by μHPLC. The flow rateis 30 μl/min with the injector, sample loop, column and transfer linesplaced in an ice bath.

A Bruker Apex II FTMS with ESI is employed for on line monitoring of thechromatographic effluent. A 1.1 sec hexapole accumulation is performedbefore ion injection, excitation and detection (m/z 350-2000). Two 256 kspectra are averaged for each stored spectrum, 128 averaged spectra areacquired per run. Resolution is 55,000 FWHM. An external calibrationwith in-source fragment ions of porcine renin substrate is performed.The trapping and excitation values are optimized to reduce variation ofmass assignment with ion population. The hexapole accumulation has theeffect of integrating the ESI ions and thus improving the measurement ofrelative abundance data. Capillary exit voltage is typically 70 V.

Spectra from each time point are analyzed. A program entitled ExPro(Poster entitled “ExchangePro: An Automated High Performance SoftwarePackage for analysis of Deuterium Exchange Mass Spectrometric DataObtained by FTMS”, presented at the 52nd annual conference of theAmerican Society for Mass Spectrometry) is used to calculate the“average mass” of the peptide's molecular ion cluster, by multiplyingthe mass times the intensity for each isotope of the cluster anddividing by the total intensity. The resulting data is plottedgraphically for each peptide, yielding a map of protection for theprotein. For all experiments, reported data is adjusted for the numberof exchangeable hydrogens in each peptide; reportedvalue=centroid/number of non-proline residues in the peptide minus one.ViewerLite 4.2 (Accelyrs Inc.) is employed to map results onto thecrystal structures.

C. Results

51 peptic peptides [encompassing amino acids 7-20, 20-27, 20-28, 28-49,50-56, 57-68, 81-87, 71-80, 91-95, 96-104, 96-105, 105-113, 109-115,114-120, 114-121, 121-129, 130-135, 136-152, 140-151, 140-152, 165-171,166-170, 172-187, 183-199, 188-198, 199-208, 218-223, 219-223, 235-238,239-247, 248-269, 258-271, 270-274, 270-276, 270-280, 281-289, 293-301,310-320, 313-320, 321-326, 326-330, 326-333, 327-332, 327-333, 331-335,331-346, 347-362, 352-362, 363-374, 374-400, 378-386, 378-387] derivedfrom the extracellular domain of NPRA are followed at each of 5 timepoints ranging from 5 to 1000 seconds. The differences in deuteriumexchange rates upon ANP and ANP+antibody binding are determined and thepeptides with the most significant changes are mapped onto a homologymodel of ANP bound to the extracellular domain of human NPRA derivedfrom the publicly available crystal structure of ligand bound rat NPRA(Ogawa, et al. (2004) J. Biol. Chem. 279:28625) The results from thebinding of 5591 are depicted in FIG. 21.

The area shaded in pink (encompassing amino acids 28-87, 96-113,293-301, 310-312, 334-335 and 352-362) represents strong protection fromexchange and that shaded in red (encompassing amino acids 7-28, 121-129,313-320, 327-333, 347-351) very strong protection. The areas shaded inred are represented by an epitope that comprises residues 7-28(NLTVAVVLPLANTSYPWSWARV) (SEQ ID NO:30), 121-129 (VKDEYALTT) (SEQ IDNO:31), 313-320 (TMEDGLVN) (SEQ ID NO:32), 327-333 (HDGLLLY) (SEQ IDNO:33) and 347-351 (VTDGE) (SEQ ID NO:34) located in the threedimensional structure of NPRA when NPRA is bound to ANP and/or BNP andthe areas shaded in pink represent epitopes that have peptide sequences28-87 (VGPAVELALAQVKARPDLLPGWTVRTVLGSSENALGVCSDTAAPLAAVDLKWEHNPA VFL)(SEQ ID NO:35), 96-113 (APVGRFTAHWRVPLLTAG) (SEQ ID NO:36), 293-301(PEYLEFLKQ) (SEQ ID NO:37), 310-312 (FNF), 334-335 (IQ), 352-362(NITQRMWNRSF) (SEQ ID NO:38).

All of the changes are manifested as decreases in the rate of exchange.The size of the area of protection (mean 1680 A² for both partners) isin the same range as that reported for other antibody-antigen pairs. Abimodal distribution is observed in peptides protected from exchange byantibody binding. Binding to one side of the homodimer is a mechanismconsistent with that observation. Interestingly the area shaded in redcorresponds with that altered upon ANP binding to the receptor and isdistant from the binding site of ANP which is in the interface betweenthe homodimers.

The sequence of the NPRA-Fc fusion protein is:

(SEQ ID NO: 45) DGTSMGNLTVAVVIPLANTSYPWSWARVGPAVELALAQVKARPDLLPGWTVRTVLGSSENALGVCSDTAAPLAAVDLKWEHNPAVFLGPGCVYAAAPVGRFTAHWRVPLLTAGAPALGFGVKDEYALTTRAGPSYAKLGDFVAALHRRLGWERQALMLYAYRPGDEEHCFFLVEGLFMRVRDRLNITVDHLEFAEDDLSHYTRLLRTMPRKGRVIYICSSPDAFRTLMLLALEAGLCGEDYVFFHLDIFGQSLQGGQGPAPRRPWERGDGQDVSARQAFQAAKIITYKDPDNPEYLEFLKQLKHLAYEQFNFTMEDGLVNTIPASFHDGLLLYIQAVTETLAHGGTVTDGENITQRMWNRSFQGVTGYLKIDSSGDRETDFSLWDMDPENGAFRVVLNYNGTSQELVAVSGRKLNWPLGYPPPDIPKCGFDNEDPACNQDHLSTLEPIGGGSGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPVNDERPLESRGPV 

Example 12 Species Cross-Reactivity

Initial species cross-reactivity studies are performed using HEK celllines overexpressing rat, canine and rhesus monkey NPRA. The rat is apreferred model due to the availability and widespread use of heartfailure models in this species, while the dog is examined due to theextensive profiling of recombinant BNP in this species. The rhesusmonkey is also a preferred model based on the availability of sequenceinformation on NPRA and its high sequence homology to human. Theaffinities of 5591 to ANP-bound cellular NPRA of these various speciesare determined using the Kinexa technology. These apparent Kd values areobtained by globally fitting the data for two antibody concentrations.The data shown below in Table 5 illustrate that 5591 binds to NPRA ofdifferent species with pM affinity. The antibody has the highestaffinity against rat NPRA followed by dog, rhesus monkey and human.

TABLE 5 Affinity of 5591 to ANP bound NPRA expressed in HEK cellsK_(d,app) (pM) 5591 ANP Human NPRA 590 Rat NPRA 13 Canine NPRA 96 RhesusNPRA 266

The effects of 5502, 5591 and 5592 on cGMP production in response tospecies appropriate ANP and BNP are also assessed and the results aresummarized in Table 6. As with human NPRA, the antibodies potentiate thecGMP production by dog and rhesus monkey NPRA in response to both ANPand BNP. With regard to rat NPRA, modest potentiation is observed withrat ANP but no shift in the dose response to rat BNP is seen in thepresence of the antibodies. FACS experiments confirm that the antibodiesbinds to rat NPRA in the presence of BNP.

TABLE 6 Potentiation of Antibody mediated cGMP responses in ANP or BNPtreated HEK NPRA cells Rat Dog Rhesus Monkey Fold shift in EC₅₀ Foldshift in EC₅₀ Fold shift in EC₅₀ IgG ANP BNP ANP BNP ANP BNP 5502 2-3None 3-5 7-10 2-8 12 5591 3 None 3-4 4-5  2-4 14 5592 5-6 None 1-4 7-102-6 16

Example 13 Pharmacokinetics Studies: ELISA for Detection of Antibody inPlasma

A sandwich ELISA is developed to determine the concentration of theantibodies in plasma. The capture antigen is ANP-bound NPRA-Fc fusionprotein and the detection antibody is either a monoclonal anti-humanIgG4 (for monkey plasma samples) or a monoclonal anti-human kappa chain(for rat and dog plasma samples) conjugated to horseradish peroxidase.Specifically, 96-well Nunc-Immuno MaxiSorp plates (Thermo FisherScientific, Rochester, N.Y.) are coated overnight with human NPRA-Fccontaining 1 nM ANP. The plates are then blocked with 3% bovine serumalbumin (BSA) in phosphate buffered saline (PBS) (BSA/PBS) for 1 hour atroom temperature. The plates are then washed three times with PBScontaining 0.1% Tween 20 (PBST). For generating the standard curve,e.g., 5591 serially diluted 1:3, from 600 ng/ml to 0.09 ng/ml in 0.5%BSA/PBS with 1 nM ANP is added to the plate and incubated for 2 hours atroom temperature. For the test samples, plasma is diluted at least 1:10in PBS with 1 nM ANP. After the 2 hour incubation, plates are washedthree times with PBST containing 0.5% BSA. To detect bound antibody,mouse anti-human IgG4-HRP or mouse anti-human kappa chain-HRP in 0.5%BSA/PBS is added and the plates are incubated at room temperature for 1hour. After the 1 hour incubation, plates are washed three times withPBST containing 0.5% BSA. One final plate wash is done using PBS only.Color is developed by adding ABTS Single Solution for 10-20 min. Platesare read at 405 nm.

Example 14 Single Dose IV and SC Antibody Pharmacokinetics in Rat

5591 is dosed at 0.3 mg/kg IV and 2 mg/kg SC to male SD rats (N=3) inphosphate buffered saline vehicle. Blood samples are collected from0-168 hr in K2-EDTA tubes and plasma samples were stored at −70° C.until analysis. The samples are analyzed by sandwich ELISA as describedabove. Pharmacokinetic parameters are summarized in Table 7. Theantibody shows low clearance and volume of distribution (Vss) and aterminal half-life of 5.8 days. The antibody is rapidly and wellabsorbed after SC dose reaching a peak at about 3 days post dose and theSC bioavailability is 81%.

TABLE 7 Pharmacokinetic parameters of 5591 in male SD rats (N = 3) aftersingle 0.3 mg/kg IV and 2 mg/kg SC doses. Dose CLp Vss AUC Cmax Tmax T½MRT F Route (mg/kg) (mL/hr/kg) (mL/kg) (μM · hr) (nM) (days) (days)(days) (%) IV 0.3 0.94 ± 0.16 165 ± 34  2.2 ± 0.3 5.8 ± 0.1 7.2 ± 0.3 SC2.0 11.6 ± 1.9 59.9 ± 0.2 3.0 ± 0.0 80.8

Example 15 Single Dose IV and SC Pharmacokinetics of Antibody in Dog

5591-IgG is dosed at 0.3 mg/kg IV to male beagle dogs (N=3) in 50 mMacetate/100 mM arginine/50 mM Tris and at the same dose SC in phosphatebuffered saline vehicle. Blood samples are collected from 0-506 hr (3weeks) in K2-EDTA tubes and plasma samples are stored at −70° C. untilanalysis. The samples are analyzed by sandwich ELISA as described above.

Pharmacokinetic parameters are summarized in Table 8. The antibody showslow clearance, low volume of distribution (Vss) approximating bloodvolume and a terminal half-life of 81 hours (˜3 days).

TABLE 8 Pharmacokinetic parameters of 5591-IgG in male beagle dog (N =3) after single 0.3 mg/kg IV and SC doses. Dose CLp Vss AUC Cmax Tmax T½MRT F Route (mg/kg) (mL/hr/kg) (mL/kg) (μg · hr) (nM) (days) (days)(days) (%) IV 0.3 0.81 ± 0.01 84 ± 15 2.5 ± 0.2 3.0 ± 0.6 4.4 ± 1.1 SC0.3 3.5 ± 0.7 9.2 ± 0.8 3.3 ± 1.2 139

Example 16 Single Dose IV and SC Pharmacokinetics of Antibodies inMonkey

5591-IgG is dosed at 0.3 mg/kg IV and SC to male cynomolgus monkeys(N=3) in phosphate buffered saline vehicle. Blood samples are collectedfrom 0-506 hr (3 weeks) in K2-EDTA tubes and plasma samples were storedat −70° C. until analysis. The samples are analyzed by sandwich ELISA asdescribed above.

Pharmacokinetic parameters are summarized in Table 9. The antibody showslow clearance, low volume of distribution (Vss) slightly higher than theblood volume of 85-90 mL/kg and a terminal half-life of 203 hr (8.5days). The antibody was slowly and well absorbed after SC dose reachinga peak at about 3 days post dose and the SC bioavailability was 145% andthe reasons for greater than 100% bioavailability are currently notknown.

TABLE 9 Pharmacokinetic parameters of BI 655,002 in male cynomolgusmonkies (N = 3) after single 0.3 mg/kg IV and SC doses. Dose CLp Vss AUCCmax Tmax T½ MRT F Route (mg/kg) (mL/hr/kg) (mL/kg) (μM · hr) (nM)(days) (days) (days) (%) IV 0.3 0.54 ± 0.02 143 ± 12.0 3.7 ± 0.2 8.5 ±0.6 10.9 ± 0.6 SC 0.3 5.4 ± 0.5 17.5 ± 1.8 2.7 ± 0.6 145

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of pharmacokineticanalyses, recombinant DNA methods, peptide and protein chemistries,nucleic acid chemistry and molecular and cellular biology describedherein are those well known and commonly used in the art.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the disclosure herein, including the appended embodiments

1. An isolated antibody or antigen-binding portion thereof thatselectively binds to an epitope located in the extracellular domain ofnatriuretic peptide receptor A (NPRA) wherein said epitope comprisesamino acids residues within a) 7-28  (SEQ ID NO: 30)(NLTVAWLPLANTSYPWSWARV); b) 28-87  (SEQ ID NO: 35)(VGPAVELALAQVKARPDLLPGWTVRTVLGSSENALGVCSDTAAP LAAVDLKWEHNPAVFL);c) 96-113  (SEQ ID NO: 36) (APVGRFTAHWRVPLLTAG); d) 121-129 (SEQ ID NO: 31) (VKDEYALTT); e) 293-301  (SEQ ID NO: 37) (PEYLEFLKQ);f) 310-312  (FNF); g) 334-335  (IQ); h) 313-320  (SEQ ID NO: 32)(TMEDGLVN); i) 327-333  (SEQ ID NO: 33) (HDGLLLY); j) 347-351 (SEQ ID NO: 34) (VTDGE);  or k) 352-362  (SEQ ID NO: 38) (NITQRMWNRSF).


2. The isolated antibody or antigen-binding portion thereof according toclaim 1, wherein the mammalian natriuretic peptide receptor A (NPRA) isa human natriuretic peptide receptor A (NPRA). 3-6. (canceled)
 7. Theisolated antibody or antigen-binding portion thereof according to claim2, wherein a ligand of NPRA is ANP or BNP. 8-10. (canceled)
 11. Theisolated antibody or antigen-binding portion thereof of claim 1, whereinthe EC₅₀ of ligand induced intracellular cGMP production is at least2-fold lower than it would be if the antibody or antigen-binding portionthereof was not present. 12-14. (canceled)
 15. The isolated antibody orantigen-binding portion thereof according to claim 1, wherein theantibody or antigen-binding portion thereof is a monoclonal antibody.16. The isolated antibody or antigen-binding portion thereof accordingto claim 1, wherein the antibody is an IgG antibody.
 17. The isolatedantibody or antigen-binding portion thereof according to claim 1,wherein the antibody is an IgG4 or an IgG1 antibody.
 18. The isolatedantibody or antigen-binding portion thereof according to claim 1,wherein the antibody or antigen-binding portion thereof is selected fromthe group consisting of: a human antibody, a humanized antibody, amurine antibody, a chimeric antibody, a peptibody, a single chainantibody, a single domain antibody, a Fab fragment, a F(ab′)₂ fragment,a Fv fragment, scF_(v) fragment and fusion proteins.
 19. An isolatedantibody or antigen-binding portion thereof that selectively binds to anepitope located in an extracellular domain of natriuretic peptidereceptor A (NPRA) wherein said epitope forms when said NPRA is bound toatrial natriuretic peptide (ANP) or brain natriuretic peptide (ANP) aand wherein said antibody is not reactive with the atrial natriureticpeptide (ANP) or brain natriuretic peptide (ANP) binding site of NPRA.20. The antibody of claim 19, wherein said antibody potentiates theactivity of said ligand or activating protein through said receptor.21-32. (canceled)
 33. An isolated antibody or antigen-binding portionthereof that selectively binds to an epitope located in an extracellulardomain of natriuretic peptide receptor A (NPRA) and competes with amonoclonal antibody comprising a VH and VL chain, each VH and VL chaincomprising hypervariable regions CDR1, CDR2 and CDR3 separated byframework amino acid sequences, the hypervariable regions having aminoacid sequences in each VH and VL wherein VH^(CDR1) of said antibody hasa sequence of SEQ ID NO:3; VH^(CDR2) of said antibody has a sequenceselected from the group consisting of SEQ ID NO:4 and 10, VH^(CDR3) ofsaid antibody has a sequence of SEQ ID NO:13, VL^(CDR1) of said antibodyhas a sequence of SEQ ID NO:14, VL^(CDR2) of said antibody has asequence of SEQ ID NO:15; and VL^(CDR3) of said antibody has a sequenceselected from the group consisting of SEQ ID NO:16, 17 and
 18. 34. Anisolated antibody or antigen-binding portion thereof according to claim33 that binds to the same epitope bound by an antibody comprising a VHand VL chain, each VH and VL chain comprising hypervariable regionsCDR1, CDR2 and CDR3 separated by framework amino acid sequences, thehypervariable regions having amino acid sequences in each VH and VLwherein VH^(CDR1) of said antibody has a sequence of SEQ ID NO:3;VH^(CDR2) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:4 and 10, VH^(CDR3) of said antibody has asequence of SEQ ID NO: 13, VL^(CDR1) of said antibody has a sequence ofSEQ ID NO:14, VL^(CDR2) of said antibody has a sequence of SEQ ID NO:15;and VL^(CDR3) of said antibody has a sequence selected from the groupconsisting of SEQ ID NO:16, 17 and
 18. 35. An isolated antibody orantigen-binding portion thereof according to claim 34 comprising a heavychain variable region (V_(H)) and a light chain variable region (V_(L)),each V_(H) and V_(L) comprising hypervariable regions CDR1, CDR2 andCDR3 separated by framework amino acid sequences, the hypervariableregions having amino acid sequences in each VH and VL chain of each VHand VL chains of: VH^(CDR1) having a sequence of SEQ ID NO:3; VH^(CDR2)having a sequence selected from the group consisting of SEQ ID NO:4 and10, VH^(CDR3) having a sequence of SEQ ID NO:13, VL^(CDR1) having asequence of SEQ ID NO:14, VL^(CDR2) having a sequence of SEQ ID NO:15;and VL^(CDR3) having a sequence selected from the group consisting ofSEQ ID NO: 16, 17 and
 18. 36-38. (canceled)
 39. An isolated nucleic acidmolecule comprising a sequence encoding an antibody or antigen-bindingportion thereof according to claim
 33. 40-45. (canceled)
 46. A vectorcomprising and capable of expressing the nucleic acid molecule accordingto claim
 39. 47. A host cell transformed with the vector of claim 46.48-49. (canceled)
 50. A pharmaceutical composition comprising thepurified antibody or antigen-binding portion thereof according to claim1 and a pharmaceutically acceptable carrier or excipient thereof. 51.(canceled)
 52. A method of producing an isolated antibody orantigen-binding portion thereof comprising the steps of culturing thehost cell according to claim 47 under suitable conditions and recoveringthe antibody or antigen-binding portion thereof.
 53. (canceled)
 54. Amethod for potentiating an apparent affinity of a ligand or anactivating protein to an extracellular domain of natriuretic peptidereceptor A (NPRA), comprising contacting NPRA with the purified antibodyor antigen-binding portion thereof according to claim 1 in the presenceof the ligand or activating protein. 55-72. (canceled)
 73. A method fortreating a disorder or condition associated with a decreased level ofcatalytic activity of natriuretic peptide receptor A (NPRA) in a subjectcomprising administering to the subject in need thereof an effectiveamount of a purified antibody or antigen-binding portion thereofaccording to claim
 1. 74. A method for treating a disorder or conditionassociated with a decreased level of catalytic activity of natriureticpeptide receptor A (NPRA) in a subject comprising administering to thesubject in need thereof an effective amount of the pharmaceuticalcomposition according to claim
 50. 75-94. (canceled)